Activating NOTCH1 Mutations Research Articles

Effect of Ferroptosis Inducers and Inhibitors on Cell Proliferation in Acute Leukemia.

Ferroptosis refers to an iron-dependent mechanism of regulated cell death that is attributable to lipid peroxidation. Ferroptosis has been documented as a therapeutic target for various solid cancers; nonetheless, its implication in leukemia remains ambiguous. Therefore, this study aimed at investigating the impact of ferroptosis inducers and inhibitors on in vitro leukemia cell line proliferation. Six leukemia cell lines, including acute myeloid leukemia (AML)-derived MV4-11, THP-1, HL-60, and U-937, and T-lymphoblastic leukemia (T-ALL)-derived Jurkat and KOPT-K1 with activating NOTCH1 mutations, were assessed. Erastin, which interrupts cystine uptake and depletes intracellular glutathione, and RAS-selective lethal 3 (RSL3), which suppresses glutathione peroxidase 4 (GPX4), were employed as ferroptosis inducers. Lipid peroxidation-arresting ferrostatin-1 and deferoxamine were used as ferroptosis inhibitors. Cells were cultured with these compounds and cell proliferation was assessed using a colorimetric assay. Additionally, signaling protein expression was monitored using immunoblotting, and the outcome of GPX4 knockdown was evaluated. Ferroptosis inducers suppressed proliferation in all cell lines except THP-1 for Erastin and THP-1 and Jurkat for RSL3. Although the ferroptosis inhibitors did not affect cell proliferation, they rescued inducer-mediated growth suppression. Ferroptosis inducers impeded MYC and cyclin D3 expression in certain cell lines and NOTCH1 signaling in T-ALL cells. GPX4 knockdown and RSL3 treatment interrupted MYC and cyclin D3 expression, respectively, in four cell lines. Ferroptosis inducers may serve as potential candidates for novel molecular therapy against AML and T-ALL.

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.

CDK12 alterations and ARID1A mutations are predictors of poor prognosis and therapeutic targets in high-grade salivary gland carcinoma: analysis of the National Genomic Profiling Database.

Due to the diversity of histopathologic types in salivary gland carcinoma, genomic analysis of large cohorts with next-generation sequencing by histologic type has not been adequately performed. We analysed data from 93 patients with salivary duct carcinoma and 243 patients with adenoid cystic carcinoma who underwent comprehensive genomic profiling testing in the Center for Cancer Genomics and Advanced Therapeutics database, a Japanese national genome profiling database. We visualised gene mutation profiles using the OncoPrinter platform. Fisher's exact test, Kaplan-Meier analysis, log-rank test and Cox regression models were used for statistical analysis. In salivary duct carcinoma, a population with CDK12 and ERBB2 co-amplification was detected in 20 of 37 (54.1%) patients with ERBB2 amplification. We identified five loss-of-function variants in genes related to homologous recombination deficiency, such as BRCA2 and CDK12. Cox survival analysis showed that CDK12 and ERBB2 co-amplification is associated with overall survival (hazard ratio, 3.597; P=0.045). In salivary duct carcinoma, NOTCH1 mutations were the most common, followed by mutations in chromatin modification genes such as KMT2D, BCOR, KDM6A, ARID1A, EP300 and CREBBP. In the multivariate Cox analysis, activating NOTCH1 mutations (hazard ratio, 3.569; P=0.009) and ARID1A mutations (hazard ratio, 4.029; P=0.034) were significantly associated with overall survival. CDK12 and ERBB2 co-amplification is associated with a poor prognosis in salivary duct carcinoma. Chromatin remodelling genes are deeply involved in tumour progression in adenoid cystic carcinoma. One such gene, ARID1A, was an independent prognostic factor. In salivary duct carcinoma and adenoid cystic carcinoma, there might be minor populations with mutations that could be targeted for treatment with the synthetic lethality approach.

Abstract 4897: PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands

Abstract Adenoid Cystic Carcinoma (ACC) is a rare but aggressive malignancy of salivary gland, associated with protracted clinical course and fatal outcome. Treatment modalities are restricted to surgery and/or adjuvant radiotherapy and patients develop recurrence and distant metastasis over time. The absence of effective systematic therapy makes it incurable in advanced stage. ACC display an overall low mutation frequency, therefore very few actionable genetic alterations critical to the ACC development have been recognized. MYB fusion/overexpression is the most frequently found genetic alteration in ACC and is present in ~70% patients. This is followed by activating NOTCH mutations, reported in ~20% patients. Despite the presence of few targetable genetic alterations, recent studies revealed substantial transcriptomic heterogeneity amongst ACC tumors suggesting the role of epigenetic alterations in ACC oncogenesis. Symmetric dimethylation is an important epigenetic mechanism that regulates mRNA splicing, transcription, translation, cell cycle and oncogenic signaling pathways. Protein Arginine Methyl Transferase 5 (PRMT5) is a predominant enzyme for symmetric dimethylation among a family of 9 methyl transferases. In was suggested that PRMT5 plays a role in regulating tumor progression via modulation of MYC signaling, cancer stemness, and a wide array of additional cellular and transcriptional pro-oncogenic processes. While inhibition of PRMTs showed anti-oncogenic response in several preclinical tumor models and a subset of patients with advanced solid malignancies (including ACC), preclinical studies investigating the effect of PRMT5 blockade in ACC remain inadequate. In part, due to the scarcity of ACC specimens and limited availability of the experimental models. PRT543 is a selective and potent small molecule PRMT5 inhibitor that specifically targets PRMT5 among 37 methyl transferases. In the present study, we have investigated the effect this novel agent using a unique collection of ACC cell lines, organoids, and patient derived xenograft mouse models. To our knowledge, this is the first study investigating the therapeutic effect of PRT543 in several preclinical models of ACC. Specifically, we found that PRT543 has potent antitumor activity in in-vitro and in-vivo models with MYB expression and activating NOTCH mutations. Further, based on these observations, we have sequenced a collection of 50 ACC tumor samples to identify the subset of patients who may potentially benefit from PRT543 treatment based on their underlying genetic signatures. This study provides evidence underscoring the role of PRMT5 signaling in ACC and supports the clinical development of PRMT5 inhibitors for this indication. Citation Format: Vasudha Mishra, Alka Singh, Xiangying Chen, Michael Korzinkin, Claudia Wing, Viktoria Sarkisova, Alexandra Ozerova, Oksana Glushchenko, Venkat Thodima, Koichi Ito, Peggy Scherle, Mark Lingen, Rifat Hasina, Alexander Pearson, Ari Rosenberg, Alex Zhavoronkov, Bruce Ruggeri, Nishant Agrawal, Evgeny Izumchenko. PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands. [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 4897.

KDM6B protects T-ALL cells from NOTCH1-induced oncogenic stress.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors. While activating NOTCH1 mutations are the dominant genetic drivers of T-ALL, epigenetic dysfunction plays a central role in the pathology of T-ALL and can provide alternative mechanisms to oncogenesis in lieu of or in combination with genetic mutations. The histone demethylase enzyme KDM6A (UTX) is also recurrently mutated in T-ALL patients and functions as a tumor suppressor. However, its gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed, suggesting it may be necessary for sustaining the disease. Here, we used mouse and human T-ALL models to show that KDM6B is required for T-ALL development and maintenance. Using NOTCH1 gain-of-function retroviral models, mouse cells genetically deficient for Kdm6b were unable to propagate T-ALL. Inactivating KDM6B in human T-ALL patient cells by CRISPR/Cas9 showed KDM6B-targeted cells were significantly outcompeted over time. The dependence of T-ALL cells on KDM6B was proportional to the oncogenic strength of NOTCH1 mutation, with KDM6B required to prevent stress-induced apoptosis from strong NOTCH1 signaling. These studies identify a crucial role for KDM6B in sustaining NOTCH1-driven T-ALL and implicate KDM6B as a novel therapeutic target in these patients.

Cellular Plasticity As Mechanisms to Escape from NOTCH1-Inhibition in T-ALL

Cellular plasticity has recently emerged as an underlying mechanism for treatment refraction in cancer. Hence, characterizing cell states and the transcriptional changes required for state transitions is critical for the development of efficient targeted therapies. Acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and adolescents that is associated with high rates of treatment failure and early relapse. T-ALL patients frequently harbor NOTCH1 activating mutations as the driving oncogene in this disease. Using T-ALL patient-derived xenograft (PDX) models carrying activating NOTCH1 mutations, we aim to u First, to determine how cell state diversity drives treatment refraction in T-ALL, we performed full-length single-cell transcriptome analysis on 3188 immune cells collected from 2 sensitive and 3 refractory PDX models. PDX models were treated with either with NOTCH inhibitor DBZ (Dipenzazepine; 10 μM/kg every other day intravenously) or vehicle. Profiled cells were collected at both a short (5 days) and a late time point (x-150 days) post-treatment to assess the effects of DBZ. Analysis of early hematopoietic and thymic precursor signatures demonstrate expression of immature hematopoietic signatures and acquisition of alternative lineage identities coexisting within the same cell, predominantly in refractory models. Analyses of pre-treatment samples indicated that disruption of the normal developmental hierarchy was associated with blockade at an early stage of T-cell commitment and can predict response to NOTCH inhibition in vivo. Upon treatment, cells underwent dramatic transcriptional reprogramming resulting in expansion of 2 distinct populations that greatly differ in both differentiation stage and developmental trajectory. One population expresses lymphoid differentiation programs, whereas the other population is enriched in genes associated with RNA processing and more immature hematopoietic signatures. Cell fate trajectories assessment using Monocle 2, revealed that consistent with their immature hematopoietic signature, refractory cells map at the start point of the differentiation trajectory, while sensitive cells clustered at the endpoint. We next sought to characterize the differentiation hierarchies that define the immature states. Developmental potential as assessed by CytoTRACE was higher in refractory cells when compared to their sensitive counterpart. To identify determinants of cell fate in a functionally relevant manner, we inferred gene regulatory network configurations using SCENIC and defined state specific regulon activity. Among the most prominently expressed regulons present in mature states, we identified transcriptional regulators involved in T-cell differentiation such as IKAROS, GATA3, and SOX5, while in immature states we observed high transcriptional regulon activity in transcription factors associated with renewal of hematopoietic progenitors such as ATF4, BCLAF1, and MYC in addition to the chromatin remodelers SMARCA4 and EZH2. To assess whether transcriptional rewiring in refractory cells results from greater chromatin accessibility, we performed single-cell ATAC sequencing (10x Genomics) on splenic cells from 1 sensitive and 1 refractory T-ALL PDX model treated with DBZ or vehicle. After integration with corresponding single-cell RNA-sequencing data, annotated ATAC profiles demonstrated greater chromatin accessibility, predominantly in active promotor and enhancer regions, in the refractory model compared to the sensitive model. Peak to gene linkage analysis revealed widespread alterations in cis-regulatory regions near genes associated with immature hematopoietic precursor signatures such as CD33. Furthermore, greater chromatin accessibility in refractory cells was associated with an increased number of enhancer loops per expressed gene as determined by H3K27ac HiChIP analysis. In conclusion, we demonstrated that presence of highly plastic cellular states defined by aberrant differentiation trajectories, distinct transcriptional circuitries, and remodeled chromatin architecture results in treatment escape in T-ALL.

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

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

Genetic heterogeneity and therapeutic target detection through microdissection in solid-type adenoid cystic carcinoma

Solid-type adenoid cystic carcinomas (ACCs) are highly aggressive and heterogeneous tumours. Because of their rarity, therapeutic strategies guided by genetic profiles based on next generation sequencing (NGS) have not been published for these tumours. Forty-nine solid-type ACCs including 43 tumours with a predominantly solid pattern, and six tumours comprising a roughly equal mixture of cribriform/tubular and solid histological forms were included in our study. The solid components from the 49 solid ACCs were enriched for mutations of genes in the NOTCH pathway (NOTCH1 61%, SPEN 24%) and chromatin remodelling pathway and the absence of myoepithelial cell differentiation. Cases with NOTCH1 mutations exhibited strong NICD expression, which was associated with poor overall and distant metastasis free survival. BRCA2 mutation and BCOR/BCORL1 mutations were observed in 20% and 18.4% of solid ACCs, respectively. In six of the solid ACCs, intratumour heterogeneity was delineated between the cribriform/tubular and solid components. NOTCH1 and FGFR2 mutations as well as NOTCH2 amplification were restricted to the solid component, indicating clonal selection within the same tumour. In two recurrent/metastatic solid ACCs, the subclones evolved in progression for local relapse and distant metastasis, although they manifested close genomic resemblance to primary tumours. Guided by the genetic profiles, the preclinical efficiency of the gamma-secretase inhibitor BMS-906024 was evaluated in patient derived xenograft models (PDXs) with activating NOTCH1 mutations and demonstrated robust antitumour effects. Our report revealed intratumour heterogeneity among solid-types within an ACC as well as the inter-tumour evolution of dominant clones among two primary and recurrent/metastatic tumours. In contrast to cribriform/tubular ACCs, solid-type ACCs should be approached with a distinct therapeutic strategy, particularly targeting NOTCH1. Microdissecting the highest grade component guided by histology is a highly recommended tumour sampling strategy and facilitates the detection of key molecular targets.

ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells

AbstractMixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

Abstract 1138: PRMT5 inhibition downregulates MYB and NOTCH1 signaling, key molecular drivers of adenoid cystic carcinoma

Abstract Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands, characterized by slow and unpredictable growth, unrelenting relapse and high rates of metastasis. Poor response to chemotherapy and targeted drugs has resulted in there being no approved therapies to date, which contributes to poor prognosis with less than a 20% 5-year survival rate in patients with high-risk ACC. A vast majority of ACC tumors are known to have recurrent chromosomal translocation t(6;9) or t(8;9) resulting in MYB/MYBL1-NFIB fusions, and high levels of MYB protein expression. Furthermore, a subset of ACC tumors expresses NOTCH1 activating mutations (20-25%) and hyperactive NOTCH signaling in both primary and recurrent/metastatic tumors. A previous report has demonstrated that an inhibitor of PRMT5 (protein arginine methyltransferase) shows favorable responsiveness in patients with advanced ACC in a phase I clinical trial. Here, we investigate mechanistic aspects as to how PRMT5 inhibition potentially regulates the unique molecular drivers of ACC tumor growth. Due to a limited number of validated in vitro cellular models, we selected a group of head and neck cancer cell lines, including those originating from salivary gland, along with MYB and NOTCH1-driven leukemia models, to evaluate the effects of PRMT5 inhibition on MYB and NOTCH regulated genes. Treatment with PRT543, a potent and selective PRMT5 inhibitor, decreased expression of MYB and its associated downstream gene GATA3, as well as ACC-related genes such as SOX4 and POU3F2. Furthermore, PRT543 downregulated MYB signaling (MYB, FOXM1, NIK and SKI) as well as MYB alternative isoforms MYB-9A and MYB-10A in MYB expressing leukemia cells. Intriguingly, treatment with PRT543 also downregulated the transcription factor, HSF4, that is uniquely linked to regulation by N-terminal truncated MYB, a variant driven by an alternative promoter (TSS2) and reported as being active in ACC tumors. In addition to regulation of MYB, we also show that PRT543 downregulates NOTCH1 expression as well as multiple NOTCH1 target genes in leukemia cells which harbor a NOTCH1 active mutation. Given these results, we investigated the ability of PRT543 to inhibit the growth of ACC PDX tumor models in vivo and demonstrate that PRT543 significantly inhibits the growth of ACC PDX tumors in vivo. We confirmed that PRT543 downregulates levels of global symmetric dimethylarginine (sDMA, a substrate of PRMT5) in the treated tumors. Further studies with PRT543 in ex vivo and in vivo ACC models are ongoing. Collectively, these findings provide a strong molecular rationale for exploring PRT543 as a potential therapeutic option for ACC tumors. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Jack Carter, Koichi Ito, Venkat Thodima, Neha Bhagwat, Joseph Rager, Nicole Spardy Burr, Jeffrey Kaufman, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition downregulates MYB and NOTCH1 signaling, key molecular drivers of adenoid cystic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1138.

Single-cell RNA-seq reveals developmental plasticity with coexisting oncogenic states and immune evasion programs in ETP-ALL

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.

Activating NOTCH1 Mutations Alter T-ALL Chemosensitivity By PREX2-AKT Pathway

Mutations on NOTCH1 gene are the most commonly found mutations in T-cell acute lymphoblastic leukemia (T-ALL) and they are reported to be favorable indicators for T-ALL patients' prognosis. However, the effects of activating NOTCH1 mutations on T-ALL's chemosensitivity have not been studied. We reported that NOTCH1 inhibition by γ-secretase inhibitors or shRNA knockdown in MOLT-3 cells could reduce the T-ALL cells' sensitivity to chemotherapeutic drugs. However, this effect was absent in Jurkat and CUTLL cells. We further demonstrated that NOTCH1 inhibition could activate the PI3K-AKT pathway in a cell specific pattern similar as their effects on chemosensitivity. RNA-seq revealed that Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is a target gene of NOTCH1 and may mediate the effects of activating NOTCH1 mutations on chemosensitivity. Consistently, we proved that overexpression of PREX2 could mimic the effects of NOTCH1 inhibition on chemosensitivity. Our study has highlighted the effects of activating NOTCH1 mutations on T-ALL's chemosensitivity by altering PREX2-AKT pathway, which may explain the favorable effects of NOTCH1 mutations on T-ALL patients' prognosis, as well as provided potential targets to alter T-ALL cells' chemosensitivity. Disclosures No relevant conflicts of interest to declare.

A Molecular Test for Quantifying Functional Notch Signaling Pathway Activity in Human Cancer.

Simple SummaryThe Notch signal transduction pathway is important for various physiological processes, including immune responses, and plays a role in many diseases, for example cancer. We have developed a new assay to quantitatively measure Notch pathway activity, and we validated it using data from various human cancer cell lines. The assay can be applied across different cell types, and offers numerous possibilities to explore the contribution of the Notch pathway to tumor formation and the stratification of cancer patients. We assessed Notch pathway activity in a cohort of T cell acute lymphoblastic leukemia (T-ALL) patient samples, and found that the pathway activity score more accurately reflects Notch pathway activity than a prediction on the basis of NOTCH1 mutations alone. Finally, we found that patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients who had T-ALL cells with higher activity.Background: The Notch signal transduction pathway is pivotal for various physiological processes, including immune responses, and has been implicated in the pathogenesis of many diseases. The effectiveness of various targeted Notch pathway inhibitors may vary due to variabilities in Notch pathway activity among individual patients. The quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. Methods: We here describe a new assay that infers a quantitative Notch pathway activity score from the mRNA levels of generally conserved direct NOTCH target genes. Following the calibration and biological validation of our Notch pathway activity model over a wide spectrum of human cancer types, we assessed Notch pathway activity in a cohort of T-ALL patient samples and related it to biological and clinical parameters, including outcome. Results: We developed an assay using 18 select direct target genes and high-grade serous ovarian cancer for calibration. For validation, seven independent human datasets (mostly cancer series) were used to quantify Notch activity in agreement with expectations. For T-ALL, the median Notch pathway activity was highest for samples with strong NOTCH1-activating mutations, and T-ALL patients of the TLX subtype generally had the highest levels of Notch pathway activity. We observed a significant relationship between ICN1 levels and the absence/presence of NOTCH1-activating mutations with Notch pathway activity scores. Patients with the lowest Notch activity scores had the shortest event-free survival compared to other patients. Conclusions: High Notch pathway activity was not limited to T-ALL samples harboring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate Notch signaling. The measured Notch pathway activity was related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than when it is predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.

The Rigor and Transparency Index Quality Metric for Assessing Biological and Medical Science Methods.

SummaryThe reproducibility crisis is a multifaceted problem involving ingrained practices within the scientific community. Fortunately, some causes are addressed by the author's adherence to rigor and reproducibility criteria, implemented via checklists at various journals. We developed an automated tool (SciScore) that evaluates research articles based on their adherence to key rigor criteria, including NIH criteria and RRIDs, at an unprecedented scale. We show that despite steady improvements, less than half of the scoring criteria, such as blinding or power analysis, are routinely addressed by authors; digging deeper, we examined the influence of specific checklists on average scores. The average score for a journal in a given year was named the Rigor and Transparency Index (RTI), a new journal quality metric. We compared the RTI with the Journal Impact Factor and found there was no correlation. The RTI can potentially serve as a proxy for methodological quality.

Ablation of the Brca1-Palb2 Interaction Phenocopies Fanconi Anemia in Mice.

Heterozygous mutations in the BRCA1 gene predispose women to breast and ovarian cancer, while biallelic BRCA1 mutations are a cause of Fanconi anemia (FA), a rare genetic disorder characterized by developmental abnormalities, early-onset bone marrow failure, increased risk of cancers, and hypersensitivity to DNA-crosslinking agents. BRCA1 is critical for homologous recombination of DNA double-strand breaks (DSB). Through its coiled-coil domain, BRCA1 interacts with an essential partner, PALB2, recruiting BRCA2 and RAD51 to sites of DNA damage. Missense mutations within the coiled-coil domain of BRCA1 (e.g., L1407P) that affect the interaction with PALB2 have been reported in familial breast cancer. We hypothesized that if PALB2 regulates or mediates BRCA1 tumor suppressor function, ablation of the BRCA1-PALB2 interaction may also elicit genomic instability and tumor susceptibility. We generated mice defective for the Brca1-Palb2 interaction (Brca1 L1363P in mice) and established MEF cells from these mice. Brca1 L1363P/L1363P MEF exhibited hypersensitivity to DNA-damaging agents and failed to recruit Rad51 to DSB. Brca1 L1363P/L1363P mice were viable but exhibited various FA symptoms including growth retardation, hyperpigmentation, skeletal abnormalities, and male/female infertility. Furthermore, all Brca1 L1363P/L1363P mice exhibited macrocytosis and died due to bone marrow failure or lymphoblastic lymphoma/leukemia with activating Notch1 mutations. These phenotypes closely recapitulate clinical features observed in patients with FA. Collectively, this model effectively demonstrates the significance of the BRCA1-PALB2 interaction in genome integrity and provides an FA model to investigate hematopoietic stem cells for mechanisms underlying progressive failure of hematopoiesis and associated development of leukemia/lymphoma, and other FA phenotypes. SIGNIFICANCE: A new Brca1 mouse model for Fanconi anemia (FA) complementation group S provides a system in which to study phenotypes observed in human FA patients including bone marrow failure.See related commentary by Her and Bunting, p. 4044.

3124 – TARGETING THE NOTCH/IL-7/SKP2 CIRCUITRY IN T-ALL

Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer. Despite marked clinical successes in the treatment of childhood T-ALL, leukemia relapse, refractory disease and induction failure (≈ 30% of patients) remain significant clinical problems, often life-threatening. Thus, new therapeutic strategies are needed. Activating Notch mutations have been identified in over 50% of the cases, placing Notch signaling as a central player in T-ALL. Notch regulates the differentiation, proliferation and survival of both, normal and malignant T-cells, however its therapeutic targeting has failed. Moreover, little is known about its downstream mediators and the crosstalk with other microenvironmental cues, like the IL-7 signaling pathway. IL-7 is an essential cytokine for normal lymphoid development, and its continuous signal can lead to T-cell neoplasms. Our laboratory has focused on study the crosstalk between these two signaling pathways in T-ALL. Here we show that primary T-ALL cells carrying Notch activating mutations require IL-7 for a robust proliferation and are still responsive to ligand-dependent Notch stimulation. We found that Notch activation hypersensitize T-ALL cells to IL-7 by mediating direct transcription of IL-7Rα and thus, up-regulating its surface expression. Notch blockade significantly inhibited IL-7-mediated proliferation of T-ALL whereas IL-7 stimulation greatly potentiated Notch signaling. Investigation the downstream mechanisms common to both pathways lead us to the identification of SKP2. We discovered that SKP2, a key cell cycle regulator for cell cycle entry associated with poor prognosis in cancers, is a downstream target of Notch playing an important role in T-ALL. We demonstrated that genetic ablation or pharmacological blockade of Skp2, significantly delayed T-ALL progression and increased survival in in vivo T-ALL models. SKP2 mediates mitogenic responses to various cytokines, and we showed that is upregulated following IL-7 stimulation in human T-ALL cells, suggesting that Notch and IL-7 signaling cooperate and converge in SKP2. In conclusion, our studies indicated a cooperative effect between an oncogenic event (Notch signaling) and microenvironmental cues (IL-7 signaling) for leukemia maintenance and progression, and identify the Notch/IL-7/SKP2 circuitry as a critical network and potential therapeutic target in T-ALL.

A New Mechanism for T-ALL Leukemogenesis: Early Expression of Alpha-Beta TCR Occurs in a Natural Subset of CD4-CD8- Double Negative (DN) Thymocytes Where MHC Engagement May Drive NOTCH Mutation and Tumor Initiation

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformed thymocytes. Most human T-ALL involves hyperactive NOTCH signaling that is often caused by activating NOTCH mutations. However, the identification of specific molecular signals that might induce or select for mutation and transformation are incompletely understood. We report that an understudied low-frequency, natural thymocyte subset expresses αβ T cell antigen receptor (TCR) earlier than most cells in mice and humans; engagement of the early αβTCR by major histocompatibility complexes (MHC) can cause outgrowth of NOTCH1 mutant clones and T-ALL leukemogenesis in a mouse model of T-ALL. Assessment of 5 recent human T-ALL cases found one to present this unique CD4-CD8- double- negative (DN) stage as the earliest identifiable developmental stage. These studies present a model of T-ALL leukemogenesis that identifies (i) a natural cell stage of origin susceptible to transformation, (ii) a matching mouse model showing that a signaling receptor (αβTCR) and its ligand (MHC) drive leukemogeneis and outgrowth of tumors bearing activating NOTCH1 mutations, and (iii) a human case that presents with a tumor consistent with this model and mechanism. In past work, the pre-TCR has been shown to impact T-ALL development in mice (Campese et al, Blood 2006), but an oncogenic role for the mature αβTCR is less well characterized and somewhat surprising. This is because, although T-ALL tumor cells may express variable levels of surface αβTCR/CD3, the earliest cell stages that are thought to transform are also thought to precede stages with αβTCR expression. Most conventional αβ thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, some thymocytes in the conventional pathway rearrange both at DN stage thus exhibiting 'precocious' αβTCR (PAT) expression. Importantly, these PAT cells are indeed part of the conventional αβ lineage, being a 'subset' only due to early αβTCR expression but without known distinction in ultimate immune function (Aifantis et al, JEM 2006). We found that ~0.01% of mouse and human thymocytes are such PAT cells at steady state. To interrogate the PAT thymocyte surface phenotype, we performed multi-parametric flow cytometry with Spanning-tree Progression Analysis of Density-normalized Events (SPADE). This revealed that PAT thymocytes constitute a DN subset that is not associated with other well-described unconventional DN thymocytes known to express αβTCR, consistent with as the expectation that PATs are part of the conventional developmental pathway. We observed that the OT1 TCR transgene is expressed in mice with parallel timing and level to the natural PAT subset, allowing use of this model to study antigen-dependent signaling and oncogenesis. In a cohort study, no T-ALL was observed in wild-type C57BL/6 or OT-1.β2M-/- mice (deficient in endogenous antigen presentation), but MHC-sufficient OT-1 mice developed PAT-stage-specific T-ALL with activating NOTCH1 mutations. Transplant experiments corroborated a requirement for antigen presentation and TCR signaling for tumor maintenance as transplanted tumors grew in MHC+ but not MHC-deficient mice. This predicted that PAT thymocytes might have an unusual ability to signal through αβTCR even without coreceptor expression. When cultured in the presence of either exogenously added β2M or antigen presenting cells, both untransformed and neoplastic PAT cells upregulated CD69 in response to the OT-1 antigenic peptide, OVA. Furthermore, ex vivo analysis of PAT cells from polyclonal C57BL/6 versus MHC-deficient mice showed intrinsic upregulation of TCR-signaling-dependent Nur77 in an MHC-dependent manner. These data revealed a unique ability of PAT cells to engage in co-receptor independent but antigen-dependent signaling. Microarray analysis showed that the gene expression profile of neoplastic PAT cells from OT-1 T-ALL most closely resembled that of conventional post β-selection DN thymocytes, in agreement with the natural PAT stage during normal T cell development. These data support a model in which transformation occurred in the naturally occurring αβ PAT thymocyte subset as cell-of-origin. Collectively, our data suggest that precocious αβTCR expression and coreceptor-independent antigen engagement can cause activating NOTCH mutation and T-ALL development. Disclosures No relevant conflicts of interest to declare.

A Test to Quantify NOTCH Pathway Activity in T Cell Acute Lymphoblastic Leukemia Patients

Background The NOTCH signaling pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis in many diseases including T-cell acute lymphoblastic leukemia (T-ALL). Over 70% of T-ALL patient samples contain mutations in NOTCH1 and/or FBXW7 that result in the activation of the NOTCH pathway. Various targeted drugs are available that inhibit NOTCH signaling, but their effectiveness varies due to variable NOTCH pathway activities among individual patients. Moreover, patients' leukemic cells that lack these mutations may still require NOTCH signaling. A quick and robust quantification of NOTCH pathway activity in primary patient samples would identify patients who could benefit from NOTCH targeted treatment. Aims In primary human T-ALL samples, we aimed to determine the NOTCH pathway activity in relation to active, intracellular NOTCH1 (ICN1) levels and in relation to NOTCH1 and/or FBXW7 mutations. Additionally, we investigated whether the NOTCH pathway activity score is more accurate than a mutation-based activity prediction. Methods Our test to assess functional NOTCH pathway activity in various cell types was applied to primary human T-ALL samples. The NOTCH test infers a quantitative NOTCH pathway activity score from mRNA levels of conserved direct NOTCH target genes based on a Bayesian network model. This model describes the causal relation between up- or downregulation of NOTCH target genes and the presence of an active or inactive NOTCH transcription complex. The Bayesian model was calibrated on publically available Affymetrix U133 Plus2.0 microarray datasets of samples with an active or inactive NOTCH pathway. Following validation on multiple cell types and malignancies, we scored NOTCH pathway activation in our well-characterized cohort of 117 T-ALL patient samples and related it to clinical and biological parameters including outcome. Results The NOTCH pathway model was calibrated using a microarray dataset containing high-grade serous ovarian cancer-which has high NOTCH activity-and normal ovarian tissue samples that lack NOTCH activity. Validation of the test using datasets from primary cells and cell lines of various origins revealed that it measures the NOTCH activity status in different cellular contexts. In primary diagnostic T-ALL samples, we observed a significant relationship between NOTCH pathway activity scores and active, intracellular cleaved NOTCH1 (ICN1) protein levels and the presence of NOTCH1-activating mutations. We next scored NOTCH pathway activity over the four T-ALL subgroups ETP-ALL, TLX, Proliferative and TALLMO. The TLX subgroup had the highest NOTCH activity levels compared to the other subgroups, consistent with the high percentage of TLX cases with NOTCH1/FBXW7 mutations. Strikingly, the significance of the correlation between ICN1 levels and NOTCH pathway activity was mainly attributed to the strong NOTCH1-activating mutations that include NOTCH1 juxtamembrane domain mutations, or hetero-dimerization mutations combined with PEST domain or FBXW7 mutations. When assessing the event-free survival and relapse-free survival curves, we observed that patients with the lowest (lower than the 25th-percentile) NOTCH pathway activity scores had the shortest event-free survival compared to the others (p<0.05, log-rank test). Summary/Conclusion High NOTCH pathway activation was mostly present in-but not limited to-T-ALL samples harboring strong NOTCH1 mutations, indicating that additional mechanisms can activate NOTCH signaling. Combined with the observation that the measured NOTCH pathway activity relates to ICN1 protein levels, this indicates that the pathway activity score more accurately reflects NOTCH pathway activity than the predicted activity based on NOTCH1 mutations alone. Disclosures Holtzer: Philips Research: Employment. Verhaegh:Philips Research: Employment. van de Stolpe:The Netherlands: Employment; Eindhoven: Employment; Philips Research: Employment.

Abstract 4885: AL101 mediated tumor inhibition in Notch mutated ACC PDX models

Abstract Goal: To evaluate Notch inhibitor monotherapy and combination (combo) therapy in Adenoid Cystic Carcinoma (ACC) Patient Derived Xenograft (PDX) models with and without activating Notch mutations (mt). ACC is a rare salivary gland malignancy with no standard of care. Chemotherapy resistance limits treatment to surgery/radiation and ~60% of patients (pts) will recur. ~22% of ACC pts have Notch activating mutations that are associated with a distinct phenotype, aggressive disease and poor prognosis. In a P1 solid tumor study, 2 ACC pts were treated with AL101, an investigational gamma secretase inhibitor (J Clin Oncol 36, 2018 abstract 2515). One pt with an NRR (Negative Regulatory Region) activating mutation had a prolonged partial response, the 2nd pt with a PEST mutation had stable disease. Both had an extended PK profile with a sustained pharmacodynamic response (>50% inhibition of HES1 in peripheral blood). These results prompted us to evaluate the effect of AL101 in tumors that harbor/lack Notch activating mutations. Design: 4 ACC PDX models were evaluated: ACCx9 (Notch1 mt: NRR activating mutation), ACCx11 (Notch1 mt, tandem duplication), ACCx6 (Notch1 wt), ACCx5M1 (Notch1 VUS, not predicted to be activating). Activated Notch1 (nuclear IHC stain) was confined to the Notch mt models. Tumors were implanted into 6-12 week old athymic nude female mice. Upon reaching 150-300 mm3 tumor volume, mice were randomized to treatment (N=5), or vehicle (N=10) arms. AL101 was dosed at 7.5 mg/kg po (days 1-4 of Q7D) as single agent or in combo with cisplatin (3 mg/kg ip; qw) or everolimus (10 mg/kg; po; qd). Results: Significant tumor growth inhibition (TGI) was seen with AL101 monotherapy compared to vehicle treatment in both Notch mt models (ACCx9; 110% TGI P<0.0001, and ACCx11; 78% TGI P=0.0441). AL101 had no significant effect on tumors lacking Notch activating mutations (ACCx6: 37% TGI P>0.99, ACCx5M1: 60% TGI P=0.26). Cisplatin and everolimus monotherapy had no significant effect on Notch mt models (cisplatin: ACCx9; 43% TGI P=0.98, ACCx11; 50% TGI P=0.99, everolimus: ACCx9; 51% TGI P=0.6, ACCx11; 54% TGI P=0.99). AL101 with cisplatin or everolimus had no further benefit in the ACCx9 model (both 106% TGI P=0.98 or P>0.99 respectively). The ACCx11 model had a non-significant benefit (104% TGI P=0.17 for cisplatin combo and 107% TGI P=0.18 for everolimus combo). Interestingly, the ACCx6 Notch wt model was sensitive to everolimus (75% TGI P=0.027) with no significant benefit of adding AL101. Conclusion: AL101 monotherapy had a significant anti-tumor effect in ACC PDX tumors with Notch activating mutations and lacked effectiveness in tumors lacking such mutations. Neither cisplatin nor everolimus monotherapy treatment had a significant effect in Notch mt models, alone or in combination with AL101. These data support the clinical development of AL101 as a targeted monotherapy for ACCs with Notch activating mutations. Citation Format: Renata ferrarotto*, Genia Alpert*, Udi Gluschnaider, Rami Rauch, Adi Mondshine, Oz Solomon, Bill Kramer, Evgeny Izumchenko, John Heymach, Andrea Vergara-Silva, Jon Aster, Matti Davis. AL101 mediated tumor inhibition in Notch mutated ACC PDX models [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 4885.

ACTIVATING NOTCH1 MUTATION IN HIGH-GRADE EVOLUTION OF ADENOID CYSTIC CARCINOMA

Objective Salivary adenoid cystic carcinoma(SACC) is identified as a tumor with biphasic differentiation of epithelial and myoepithelial cells, showing tubular, cribriform and solid subtypes. The solid subtype is considered as high-grade with more aggressiveness and poorer prognosis. However, the molecular mechanism remains unknown. The aim of this study was to identify the clinicopathological characteristics of high-grade SACC, to clarify the molecular mechanism underline its distinct characteristics, and hopefully to explore potential molecular targets for SACC therapy. Study design Activated Notch1 (NICD) and myoepithelial cell markers were used for immunohistochemistry in 119 SACCs including 59 cribriform-tubular and 60 solid subtypes. Notch1 mutations were analyzed by DNA sequencing in all the SACC cases. The effect of activating NOTCH pathway on the biological behavior of SACC cell lines was investigated with transfection and functional studies. Results Notch1 mutations in the negative regulatory region and Pro-Glu-Ser-Thr–rich domains were identified in 26 of 119 patients with SACC, and 24 (92%) of 26 Notch1 mutant cases were predicted to be activating with NICD positive, with 2 cases predicted to be inactivating with NICD negative. Most (23/24, 96%) cases with activating Notch1 mutations were high-grade solid SACCs. Meantime, only 17 (18%) of 93 NOTCH1 wild-type tumors stained positive, and 16 of 17 tumors with NICD positive were high-grade solid subtypes. Furthermore, high-grade solid SACCs showed dramatically decreased short-term survival, tended to suffer bone invasion and metastasis, and presented NICD positive and myoepithelial cell markers negative simultaneously. Transfection and functional studies showed forced NICD expression promoted high level of proliferation and migration in SACC cells. Conclusions Our findings showed activating Notch1 mutations were related to the loss of myoepithelial differentiation in high-grade SACCs and might contribute to higher proliferation and worse outcome in high-grade tumors. Targeting the Notch signaling pathway in high-grade SACCs may provide therapeutic benefits.