Cell-based High-throughput Assay Research Articles

Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Abstract A095: The paradox-breaker BRAF inhibitor plixorafenib (FORE8394) efficiently inhibits non-V600 mutations and fusions

Abstract The use of several BRAF inhibitors to target BRAF V600 (Class I BRAF mutations) mutated tumors has shown clinical benefit in multiple types of cancer. Nonetheless, V600 mutations comprise about 40% of the BRAF-altered patient population, and the remainder of the BRAF mutated patients are in need of novel targeted therapies. Clinical trials aimed at evaluating the effect of these inhibitors in non-V600 patients, mostly in combination with a MEK inhibitor, have been performed with mixed results. However, the experimental sensitivity to a wide range of class 2 (RAS independent non-V600 including BRAF fusions) or class 3 (RAS dependent non-V600) alterations to different types of BRAF inhibitors has not been reported. Here we tested the efficacy of plixorafenib (a novel paradox braker BRAF inhibitor) compared with several BRAF inhibitors in BRAF V600 and non-V600 alterations. We implemented a high-throughput cell-based functional assay to profile the response of different BRAF variants to plixorafenib (paradox breaker), vemurafenib (a first-generation inhibitor) and tovorafenib (a pan-RAF inhibitor). This method quantifies MAPK signaling pathway activation using fluorescent imaging coupled with image analysis of cells expressing the mutated protein together with a fluorescently labeled ERK2 as a signaling pathway reporter. We also validated a subset of the findings using standard western blot and cell viability experiments. Comparing the response of 4 Class I, 26 Class II and 94 BRAF fusions to plixorafenib and vemurafenib Results showed that Plixorafenib was more potent in inhibiting the majority of BRAF alterations tested, i.e., a lower IC50 value was measured for plixorafenib over vemurafenib. Furthermore, of the 94 BRAF fusions proteins tested, over 75% showed at least a 50% inhibition over the concentrations tested, while less than 20% were inhibited by vemurafenib. A smaller cohort of 23 BRAF alterations was tested with the investigational pan-RAF inhibitor tovorafenib. The IC:50 values for the Class I and II BRAF alterations tested were higher with tovorafenib compared with plixorafenib, and 6 of 13 BRAF fusions tested were resistant to tovorafenib and sensitive to plixorafenib. In conclusion, we show here that plixorafenib inhibits a wide range of V600 and non-V600 BRAF alterations, more potently compared with first generation BRAFi (vemurafenib) and a pan-RAF inhibitor (tovorafenib). Importantly, this effect has the potential to overcome an unmet clinical need in cancers with non-V600 alterations. Citation Format: Limor Cohen, Natalie Filippov-Levy, Hana Billauer, Lea Birnbaum, Sara Gasasa, Roey Maor, Yakir Amrusi, Reham Abu-Liel, Eden Goldfarb, Gabi Tarcic. The paradox-breaker BRAF inhibitor plixorafenib (FORE8394) efficiently inhibits non-V600 mutations and fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A095.

Abstract A029: Treatment-Specific Immune Phenotypes Identified by nELISA High-Throughput Proteomics Reveal Actionable Insights for Drug Discovery

Abstract Understanding tumor immunology requires high content tools that can capture the complex microenvironment, as well as high-throughput cell-based assays to rapidly screen compounds, antibodies, or cell therapies. Unfortunately, proteomics tools to investigate interactions between cancer and immune cells compromise either content or cost, limiting access to phenotypic data. To overcome this issue, we developed the nELISA: a high-throughput miniaturized ELISA quantifying 191 cytokines, chemokines, proteases and growth factors, at 10x-reduced cost compared to previous tools, and applied it to cell based models to demonstrate its ability to characterize immune phenotypes in co-culture systems. We ran the largest PBMC secretome screen to date, in which ~10,000 PBMC samples were treated with various inflammatory stimuli, and were further perturbed with a selected library of 80 recombinant protein “perturbagens”. 191 secreted proteins were profiled in all samples, resulting in ~2M datapoints. The nELISA profiles were able to capture phenotypes associated with specific stimulation conditions, individual donors, and potent cytokine perturbagens. By compensating for stimulation and donor differences, we clustered perturbagens according to their effects on PBMC secretomes. As expected, perturbagens such as IFN gamma and IL-4 led to well-established Th1 or Th2 responses, respectively, and clustered with perturbagens involved in these phenotypes. Novel phenotypic effects were also identified, such as distinct responses to the near identical CXCL12 alpha and beta isoforms. Interestingly, we observed important similarities between PBMC responses to the cytokine drugs IFN beta and IL-1 Receptor antagonist, supporting the use of the latter as a replacement for the former in certain indications. These findings highlight the ability of the nELISA to capture actionable insights from high-throughput screens, and demonstrate its applicability to SAR studies and drug repurposing screens. Thus, the nELISA is a powerful tool for immunotherapy drug discovery, and we will expand upon its use for target identification, in vitro pharmacology, predicting patient response to therapy, as well as characterizing the potential of iPSC- or donor-derived material for cell therapy. Citation Format: Nathaniel Robichaud, Grant Ongo, Ivan Teahulos, Woojong Rho, Milad Dagher. Treatment-Specific Immune Phenotypes Identified by nELISA High-Throughput Proteomics Reveal Actionable Insights for Drug Discovery [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A029.

Advancing CAR-T Therapy in Acute Lymphoblastic Leukemia: Multi-Omic Analyses of CD19-Directed CAR-T Cells Enabled By an Ex Vivo Co-Culture Platform

Background: Acute Lymphoblastic Leukemia (ALL) has witnessed remarkable progress in the treatment of patients with Chimeric Antigen Receptor T-cell (CAR-T) therapies, leading to high response rates and durable remissions. However, challenges persist for patients who do not respond to CAR-T treatment, experience relapse, or encounter adverse events such as cytokine release syndrome. Overcoming these challenges necessitates a deeper understanding of mechanisms and the development of advanced technologies and assays. This study focuses on harnessing the potential of an innovative microfluidic platform for high-throughput cell-based assays, enabling multi-omics and functional analysis using a minimal amount of primary patient cells. By utilizing this platform, we aim to gain insight into the dynamics of primary patient material by evaluating responses to aid in selection of promising CAR-T enhancements or combination therapies designed to overcome mechanisms of CAR-T failure or relapse to improve clinical outcomes. Methods: For the cytotoxicity assay, we utilized autologous or allogeneic pairs of CD19-directed CAR-T cells and T-cell negative fractions from ALL patients' blood apheresis samples, containing CD19-expressing cancer cells. A negative control was established using untransduced T-cells. To ensure high cell viability and purity, the cells underwent careful processing and were labeled with long-lived dyes for tracking. Multiple cell suspensions with precise Effector to Target to Negative Fraction ratios were prepared. Only a few thousand cells from each coculture suspension were seeded in microfluidic culture chambers, which required no fluidic control system. Experiments were conducted for up to 96 hours with media replacement every 24 hours. At each endpoint, media was collected for cytokine quantification, and cells were stained for live/dead counting as well as quantification of intracellular or surface marker expression by imaging. After image acquisition, cells were collected for RNA extraction and gene expression analysis. Results: Results demonstrated that the developed co-culture platform allows precise control over culture conditions, ensuring culture of accurate ratios of highly complex cell populations. The viability of primary cells in the device guaranteed functional relevance of the cytotoxicity assays for up to a 96-hour timepoint. Strong CAR-T-mediated cell killing was observed, with a dose-dependent effect depending on the co-culture's relative CAR-T content. Less than 50% cell survival was observed in cocultures with Effector to Target ratio 1:1, as compared to the untransduced T-cell control. Furthermore, strong CAR-T activation was observed with over 60% of CART expressing CD25 and CD69 surface markers, as measured by imaging. A multi-color staining panel allowed for precise monitoring of changes over time within the assay of immune cell populations, including cancer cells, monocytes/macrophages, and NK cells, among others. Cytokine analysis revealed an increased secretion of IL-2, TNFa, IFNg, and Granzyme B in coculture with CAR-T cells compared to untransduced T-cells, with a dose-dependent effect based on the CAR-T cell ratio. Finally, RNA isolated from remaining cells at the end of the assay was of high quality with yields sufficient to enable transcriptome analysis for each treatment condition. Discussion: This study contributes to the development of innovative multiplexed high throughput strategies for in vitro functionality assessments in cell therapy, including combination studies and deeper mechanistic understandings. The platform's ability to generate fully integrated, multi-omics readings from complex allogeneic and autologous cocultures of CAR-T and patient cells offers a new frontier in early discovery and translational cancer research. This opens new possibilities for drug discovery, drug screening, and disease modeling, ultimately advancing our understanding of complex biological processes and accelerating therapeutic development. Future directions include exploring mechanisms of resistance and sensitivity, including donor-specific effector cell functionality, combination studies, impacts of immunosuppressive cell types, patient heterogeneity of target antigen expression or other co-stimulatory molecules, and evaluation of product potency or fitness of incoming apheresis material.

High-Throughput cell-based immunofluorescence assays against influenza

A rapid drug discovery response to influenza outbreaks with the potential to reach pandemic status could help minimize the virus's impact by reducing the time to identify anti-influenza drugs. Although several anti-influenza strategies have been considered in the search for new drugs, only a few therapeutic agents are approved for clinical use. The cytopathic effect induced by the influenza virus in Madin Darby canine kidney (MDCK) cells has been widely used for high-throughput anti-influenza drug screening, but the fact that the MDCK cells are not human cells constitutes a disadvantage when searching for new therapeutic agents for human use. We have developed a highly sensitive cell-based imaging assay for the identification of inhibitors of influenza A and B virus that is high-throughput compatible using the A549 human cell line. The assay has also been optimized for the assessment of the neutralizing effect of anti-influenza antibodies in the absence of trypsin, which allows testing of purified antibodies and serum samples. This assay platform can be applied to full high-throughput screening campaigns or later stages requiring quantitative potency determinations for structure-activity relationships.

Chemically induced reprogramming to reverse cellular aging.

A hallmark of eukaryotic aging is a loss of epigenetic information, a process that can be reversed. We have previously shown that the ectopic induction of the Yamanaka factors OCT4, SOX2, and KLF4 (OSK) in mammals can restore youthful DNA methylation patterns, transcript profiles, and tissue function, without erasing cellular identity, a process that requires active DNA demethylation. To screen for molecules that reverse cellular aging and rejuvenate human cells without altering the genome, we developed high-throughput cell-based assays that distinguish young from old and senescent cells, including transcription-based aging clocks and a real-time nucleocytoplasmic compartmentalization (NCC) assay. We identify six chemical cocktails, which, in less than a week and without compromising cellular identity, restore a youthful genome-wide transcript profile and reverse transcriptomic age. Thus, rejuvenation by age reversal can be achieved, not only by genetic, but also chemical means.

High-Throughput Microscopy Characterization of Rare LDLR Variants

Familial hypercholesterolemia (FH) is the most common inherited life-threatening disorder of lipid metabolism. Early diagnosis and treatment are the key to reduce the cumulative life-long cardiovascular burden of patients with FH. The high number of LDLR variants described as variants of unknown significance is the largest obstacle to achieve a definitive FH diagnosis. This study established a time- and cost-effective high-throughput cell-based assay to functionally profile LDLR variants, which allowed us to discriminate disruptive rare variants from silent ones. This work generated a valuable resource for systematic functional characterization of LDLR variants solving 1 of the major issues to achieve a definitive FH diagnosis.

A novel cell-based screen identifies chemical entities that reverse the immune-escape phenotype of metastatic tumours.

Genetic and epigenetic events have been implicated in the downregulation of the cellular antigen processing and presentation machinery (APM), which in turn, has been associated with cancer evasion of the immune system. When these essential components are lacking, cancers develop the ability to subvert host immune surveillance allowing cancer cells to become invisible to the immune system and, in turn, promote cancer metastasis. Here we describe and validate the first high-throughput cell-based screening assay to identify chemical extracts and unique chemical entities that reverse the downregulation of APM components in cell lines derived from metastatic tumours. Through the screening of a library of 480 marine invertebrate extracts followed by bioassay-guided fractionation, curcuphenol, a common sesquiterpene phenol derived from turmeric, was identified as the active compound of one of the extracts. We demonstrate that curcuphenol induces the expression of the APM components, TAP-1 and MHC-I molecules, in cell lines derived from both metastatic prostate and lung carcinomas. Turmeric and curcumins that contain curcuphenol have long been utilized not only as a spice in the preparation of food, but also in traditional medicines for treating cancers. The remarkable discovery that a common component of spices can increase the expression of APM components in metastatic tumour cells and, therefore reverse immune-escape mechanisms, provides a rationale for the development of foods and advanced nutraceuticals as therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Abstract 2753: Engineering patient-derived tumors to enable high-throughput screening: Immuno-oncology workflows

Abstract Aim/Introduction: In recent years, cancer immunotherapy has become one of the fastest-growing areas in cancer research. Selecting suitable and cost-effective experimental models for developing and validating immunotherapies is one of the major obstacles researchers face today. To overcome this, patient-derived tumor models are of increasing interest because they can better recapitulate many of the properties and the heterogeneity exhibited by the tumor microenvironment at a relatively low cost. Hereby, we propose a high throughput screening platform for an effective and efficient evaluation of cancer immunotherapies in patient-derived tumor models. Methods: Tumor models were established in vitro from patient-derived tumor biopsies. Established tumoroids were engineered using a luciferase-green fluorescent protein (GFP) lentivirus to generate a reporter pool. Transduced pools were enriched for GFP via flow cytometry and characterized using RNA/scRNA-seq and biomarker-based sequencing. Natural Killer (NK) cells were co-cultured with the enriched pool in various effector-to-target ratios and recorded using a live cell imaging and analysis platform. Cytotoxicity and cell health were measured by GFP intensity, luciferase activity, and caspase-based live staining. Results: A patient-derived tumoroid reporter pool was successfully generated through GFP enrichment using a flow cytometer. The killing efficiency of immune cells with various effector(E) to target(T) ratios has been successfully captured in a ratio-dependent manner via the live cell imaging and analysis platform. NK cell-mediated cytotoxicity was successfully measured through GFP intensity, luciferase, and caspase activity. Conclusions: Traditional cell line generation can be used in patient-derived tumoroid models to generate enriched reporter cell pools without selection pressure. Outside of establishing screening platforms, scientists can use this approach to efficiently engineer patient-derived tumoroid models to meet their specific research goals. Here, we used the reporter pool to develop a multiplex-killing assay to measure cell viability and toxicity. This platform can be used in a variety of immune cell workflows, providing a method that can predict tissue-specific responses, and evaluate solid tumor immunotherapies in high throughput cell-based assays. Citation Format: Andrew Tsao, Xiaoyu Yang, Garrett Wong, Vivek Chandra, Jacob Delgadillo, Lindsay Bailey Steinitz, Brittany Balhouse, Colin Paul, Jakhan Nguyen, Sybelle Djikeng, Shyanne Salen, Jason Sharp, Matt Dallas, David Kuninger. Engineering patient-derived tumors to enable high-throughput screening: Immuno-oncology workflows [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 2753.

Abstract 5526: State-of-the-art biobanking of gastroesophageal adenocarcinoma samples: Integrating non-viable biospecimens with 3D and 2D cell models and their characterization, validation, and utilization of cell models for precision oncology

Abstract Traditionally, biobanking platforms have collected and stored non-viable biological specimens such as serum, plasma, and fresh-frozen or formalin-fixed paraffin-embedded (FFPE) tissues. These constitute key resources for clinical and contemporary genomics, transcriptomics, and proteomics studies. However, such specimens cannot be used for studies involving drug testing, high throughput target validation, and implementation for personalized medicine. Next-generation biobanking strategies rectify this issue by combining the collection of non-viable samples from patients with the propagation of viable tissue fractions as in vitro 2D and 3D cell models or in vivo xenograft models. Such cutting-edge biobanking strategies enable downstream cell-based high-throughput functional assays promoting the discovery of therapeutic targets or assessing treatment responses and resistance to treatment. Here, we provide comprehensive details of our establishment of a state-of-the-art biobanking platform for gastroesophageal adenocarcinoma (GEA) samples (n=389). Our approach opens new directions for understanding disease biology and conducting translational cancer studies. Patient-derived 2D cells (n=376), organoids (PDOs) (n=185), and xenografts (PDXs) (n= 99) included in our pipeline retain crucial features of the original human tumors, serving as valuable tools for clinical and experimental analyses in the context of precision oncology. We also discuss the entire approach of next-generation biobanking and emphasize the importance of integrating the propagation of PDOs and PDXs simultaneously. In addition, we explain each protocol optimized to propagate patient tissue-derived cell models. We validate that these models recapitulate tissue heterogeneity and are relevant preclinical models. Taken together, we described each approach used to develop one of the largest next-level biobanks for GEA. Citation Format: Mingyang Iris Kong, Sanjima Pal, Julie Berube, France Bourdeau, Betty Giannias, Nicholas Bertos, Veena Sangwan, Lorenzo Ferri. State-of-the-art biobanking of gastroesophageal adenocarcinoma samples: Integrating non-viable biospecimens with 3D and 2D cell models and their characterization, validation, and utilization of cell models for precision oncology. [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 5526.

Antiepileptic Drugs Modulate Alzheimer-Related Tau Aggregation in a Neuronal Activity-Independent Manner

Introduction: A rapidly increasing number of patients with dementia present a serious social problem. Recently, the incidence of epilepsy in patients with Alzheimer’s disease (AD) is increasing, drawing attention to the pathological relationship between the two conditions. Clinical studies have suggested the protective action of antiepileptic agents on dementia; however, the underlying mechanism remains unknown. We evaluated the effects of multiple antiepileptic drugs using tau aggregation assay systems to determine the effects of antiepileptic agents on tau aggregation, a major neuropathological finding associated with AD. Methods: We evaluated the effects of seven antiepileptic agents on intracellular tau aggregation using a tau-biosensor cell-based high-throughput assay. Next, we tested these agents in a cell-free tau aggregation assay using thioflavin T (ThT). Results: The assay results revealed that phenobarbital inhibited tau aggregation, whereas sodium valproate, gabapentin, and piracetam promoted tau aggregation. In the cell-free tau aggregation assay using ThT, we confirmed that phenobarbital significantly inhibited tau aggregation. Conclusion: Antiepileptic drugs may modify the tau pathology in AD in a neural activity-independent manner. Our finding may provide an important insight into the optimization of antiepileptic drug therapy in older adults with dementia.

Inhibitors of One or More Cellular Aurora Kinases Impair the Replication of Herpes Simplex Virus 1 and Other DNA and RNA Viruses with Diverse Genomes and Life Cycles.

We utilized a high-throughput cell-based assay to screen several chemical libraries for inhibitors of herpes simplex virus 1 (HSV-1) gene expression. From this screen, four aurora kinase inhibitors were identified that potently reduced gene expression during HSV-1 lytic infection. HSV-1 is known to interact with cellular kinases to regulate gene expression by modulating the phosphorylation and/or activities of viral and cellular proteins. To date, the role of aurora kinases in HSV-1 lytic infection has not been reported. We demonstrated that three aurora kinase inhibitors strongly reduced the transcript levels of immediate-early (IE) genes ICP0, ICP4, and ICP27 and impaired HSV-1 protein expression from all classes of HSV-1, including ICP0, ICP4, ICP8, and gC. These restrictions caused by the aurora kinase inhibitors led to potent reductions in HSV-1 viral replication. The compounds TAK 901, JNJ 7706621, and PF 03814735 decreased HSV-1 titers by 4,500-, 13,200-, and 8,400-fold, respectively, when present in a low micromolar range. The antiviral activity of these compounds correlated with an apparent decrease in histone H3 phosphorylation at serine 10 (H3S10ph) during viral infection, suggesting that the phosphorylation status of H3 influences HSV-1 gene expression. Furthermore, we demonstrated that the aurora kinase inhibitors also impaired the replication of other RNA and DNA viruses. These inhibitors significantly reduced yields of vaccinia virus (a poxvirus, double-stranded DNA, cytoplasmic replication) and mouse hepatitis virus (a coronavirus, positive-sense single-strand RNA [ssRNA]), whereas vesicular stomatitis virus (rhabdovirus, negative-sense ssRNA) yields were unaffected. These results indicated that the activities of aurora kinases play pivotal roles in the life cycles of diverse viruses. IMPORTANCE We have demonstrated that aurora kinases play a role during HSV-1 lytic infection. Three aurora kinase inhibitors significantly impaired HSV-1 immediate-early gene expression. This led to a potent reduction in HSV-1 protein expression and viral replication. Together, our results illustrate a novel role for aurora kinases in the HSV-1 lytic cycle and demonstrate that aurora kinase inhibitors can restrict HSV-1 replication. Furthermore, these aurora kinase inhibitors also reduced the replication of murine coronavirus and vaccinia virus, suggesting that multiple viral families utilize the aurora kinases for their own replication.

Assay Development and Small Molecule Screens for Inhibitors of an RNA Binding Protein Implicated in Hematologic Malignancies

RNA-binding proteins (RBPs) play a critical role in regulating RNA splicing, polyadenylation, and translation. RBPs typically contain discrete RNA-binding domains (RBDs) that bind to specific nucleotide motifs. Given their important role in regulating gene expression, RBPs are often mutated or aberrantly expressed in a variety of malignancies, particularly hematological cancers. As a result, RBPs appear to be excellent therapeutic targets. However, given that most RBPs lack enzymatic activity, it has been challenging to develop targeted therapies. Over the past several years we and others have used genetic and molecular biology approaches to understand the consequence of aberrant expression of the poly-C RBP, heterogeneous ribonucleoprotein K (hnRNP K), and determine the mechanisms by which it drives hematologic malignancies (Cancer Cell, 2015; NEJM, 2013, JNCI, 2020; and BioRxiv, 2021). To develop a pharmacologic strategy to target hnRNP K's RNA binding activities, we developed a robust fluorescent anisotropy (FA) assay to evaluate hnRNP K's ability to bind target transcripts that are critical for tumorigenesis; such as, BRD4, MYC, RUNX1, and 7SK. We next performed small molecule screens using ~80,000 compounds and identified 34 compounds that inhibited hnRNP K's ability to bind RNA targets. To gain a deeper understanding of whether these compounds directly interact with hnRNP K to block its RNA binding functions, we performed SYPRO-orange thermal shift assays. Here we identified compounds that bound hnRNP K directly and altered hnRNP K thermostability. To evaluate association rate, dissociation rate, and KD constants, we next performed SPR assays. These experiments identified compounds bound hnRNP K with affinities between (350nM and 20uM). We next performed cell-based studies, such as CETSA. Here we identified compounds that crossed the plasma membrane, directly engaged hnRNP K in vivo, and significantly stabilized hnRNP K. Molecular analyses revealed that cells treated with these lead compounds have reduced c-Myc expression, a direct mRNA target of hnRNP K. To develop a granular understanding of how these hnRNP K-small molecule interactions impact growth and cell survival, we performed high-throughput cell-based assays. These studies demonstrated that cells with inducible hnRNP K overexpression were sensitive to these small molecules. To begin medicinal chemistry approaches and to understand each compound's chemical requirements for binding to hnRNP K, we scanned molecules for reactive moieties. Here, we identified two compounds possessing amide groups within two carbons of either a furan or pyrrole moiety. These compounds, #20 and #23, respectively, were then chemically modified and FA assays were repeated in the presence of target mRNAs. Here, we observed that both the furan and isoxazole ring of compound #20 and pyrrole rings of compound #23 are critical for hnRNP K interactions, while the cyclopropane moiety on compound #23 is dispensable. Further analyses revealed additional structural analogs with either increased or decreased affinities and these results have been critical to our understanding of the importance of each moiety. Currently, we are using cryo-EM to evaluate positional interactions between hnRNP K (hnRNP K's RBDs) and these small molecules. These results will be used to further our medicinal chemistry approaches to generate more potent analogs. Collectively, our results demonstrate that hnRNP K functions can be directly inhibited by small molecules that block its RNA binding functions and demonstrates that the development of effective therapies for RBPs is possible. 1. hnRNP K Is a Haploinsufficient Tumor Suppressor that Regulates Proliferation and Differentiation Programs in Hematologic Malignancies. Cancer Cell, 2015. 28(4): p. 486-499. 2. Cancer Genome Atlas Research Network, Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013 368(22):2059-74. 3. Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas. JNCI: Journal of the National Cancer Institute, 2020. 4. Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferative disease in mice via altered Runx1 splicing. BioRxiv. doi: https://doi.org/10.11.02.05.429385, 2021 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Screening of SARS-CoV-2 antivirals through a cell-based RNA-dependent RNA polymerase (RdRp) reporter assay.

COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose an international public health threat and thus far, has resulted in greater than 6.4 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast-spreading COVID-19 variants that may elude vaccine efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through a luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5′CT, and dasabuvir. Dasabuvir (an FDA-approved drug) exhibited promising RdRp inhibitory activity among these inhibitors. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with EC50 values 9.47 μM and 10.48 μM, for USA-WA1/2020 and B.1.617.2 variants, respectively. Our results suggest that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. Importantly, this system provides a robust, target-specific, and high-throughput screening compatible (z- and z’-factors of >0.5) platforms that will be a valuable tool for screening SARS-CoV-2 RdRp inhibitors.

Abstract 1162: A systematic analysis of RET mutations and their sensitivity to different RET inhibitors

Abstract The RET oncogene is a receptor tyrosine kinase, mainly expressed in neural crest-derived tissues, that plays a role in cell growth and differentiation. Since the first identification of inherited mutations in RET as the cause of Multiple endocrine neoplasia type 2 (MEN2), somatic RET alterations have been identified as actionable drivers in subtypes of lung and thyroid, among other cancer types. This enabled the development of several RET targeted treatments, such as pralsetinib and selpercatinib, showing meaningful clinical benefit. To understand the full scope of RET alteration frequency and heterogeneity we used the AACR-GENIE database. We found that the frequency of RET alterations pan-cancer is 3%, of which two-thirds are missense mutations. The major cancer types with RET alterations are medullary thyroid, papillary thyroid and NSCLC, as expected, but also breast, colorectal and melanoma. An important distinction between NSCLC and thyroid cancer compared to colorectal, breast and melanoma is the fraction of missense vs. fusion events, with 30%-40% of samples in the former group containing RET fusions. This is in contrast with >80% of samples in the latter group containing RET missense mutations. Moreover, the heterogeneity of RET alterations is large, with over 1,000 unique types of missense mutations identified. The vast majority of mutations are classified as variants of uncertain significance (VUS). To better understand the potential activity of these mutations, we assayed over 200 different missense mutations and fusions using a high-throughput cell-based functional assay. Results were analyzed using a novel machine learning model consisting of a multi-scale deep convolutional neural net, followed by a tree-based regressor. Our results show that the assay correctly predicted the activity of both known activating alterations as well as of loss of function mutations. Interestingly, only ~3% of over 150 VUS tested were found to be activating. Furthermore, we show that the active alterations are sensitive to the FDA-approved targeted agents pralsetinib and selpercatinib, with varying IC50 values. Taken together, these results uncover the full extent of actionable RET alterations, differentiating between active and non-active variants, as well as the effectiveness of RET inhibitors. Citation Format: Shay Rotkopf, DIkla Haham, Natalie Fillipov-Levy, Lea Birnbaum, Elinor Dehan, Ilona Kifer, Zohar Barbash, Gabi Tarcic. A systematic analysis of RET mutations and their sensitivity to different RET inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1162.

Abstract 65: A novel tool for functional characterization of mutations and their response to targeted therapy

Abstract Genomic profiling of tumors has uncovered staggering amounts of novel alterations, termed variants of uncertain significance (VUS), even in well-studied oncogenes. Strikingly, as the number of sequenced samples increases so does the number of VUS. While the study of alterations identified as VUS has shown that some are inconsequential, it has also been shown that many can cause protein activation and are involved in oncogenicity and resistance to therapy or might serve as novel drug targets. We developed a high-throughput cell-based assay that allows functional annotation of VUS and their sensitivity to different inhibitors. The system quantifies signaling pathway activation using fluorescent imaging coupled with image analysis of cells expressing the mutated protein together with a fluorescently labeled signaling pathway reporter. We show that this method accurately identifies known driver mutations in 10 different oncogenes, including receptor tyrosine kinases and signaling proteins, spanning the four major signaling pathways involved in cancer. We then deployed this system to characterize over 100 different VUS in these genes. We also show that the method correctly recapitulates differential sensitivity of cKIT and EGFR mutations to different inhibitors. Finally, we highlight the importance of concurrent mutations in KRAS which abrogate the response to EGFR inhibitors. Altogether, this system provides significant information both for development of new targeted agents as well as tailoring of optimal treatment for patients. Citation Format: Zohar Barbash, DIkla Haham, Natalie Fillipov-Levy, Limor Cohen, Lea Birnbaum, Shay Rotkopf, Ilona Kifer, Gabi Tarcic. A novel tool for functional characterization of mutations and their response to targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 65.

An Active Learning Framework Improves Tumor Variant Interpretation.

A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.

Identification of a Subtype-Selective Allosteric Inhibitor of GluN1/GluN3 NMDA Receptors.

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 μM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.

HGG-44. Unraveling and Targeting the stem-regulatory network driving invasion in Diffuse hemispheric glioma, H3G34-mutant

Diffuse hemispheric glioma H3G34-mutant (DHG-G34) is a pediatric-type high-grade glioma affecting children and young adults. Despite surgery and radio/chemotherapy, patients have a dismal prognosis. The intratumoural heterogeneity and the high infiltrative nature of DHG-G34 cells limit the development of effective therapies. Analysing single-cell RNA sequencing data from a publicly available dataset, we identified a large and distinct sub-population of cells displaying high stem and low differentiation marker expression levels. Gene ontology analyses revealed a gene signature related to cell migration/invasion. This observation is supported by our data on in vitro 3D invasion assay and in vivo orthotopic xenograft models, showing that DHG-G34 disseminating cells are characterised by high expression level of the stem-cell marker NESTIN and low expression level of the differentiation marker GFAP. Following these findings, we developed high-throughput cell-based assays with the aim to screen a library of 1300 FDA-approved compounds and identify drugs able to induce DHG-G34 cell differentiation and inhibit their invasive phenotype. The screen, a co-immunofluorescence assay for NESTIN and GFAP, followed by dose response assays on 3D growth and 3D invasion, led to the identification of 3 FDA-approved drugs, the MEK inhibitor Cobimetinib and 2 HMG-CoA reductase inhibitors, Rosuvastatin and Pitavastatin. These 3 drugs potently induced cell differentiation (decreased Nestin and increased GFAP expression) and inhibited invasion with minimal effect on the proliferation of our DHG-G34 cell line. We are currently extending these findings to additional patient-derived DHG cell lines and we are using these drugs and different omics and imaging technologies to characterize the regulatory networks associated to DHG-G34 stemness, (de)-differentiation and invasiveness. Our work may lead to the identification of new therapeutic approaches for targeting the stem/invasive properties of these aggressive diseases.

High throughput screening for compounds to the orphan nuclear receptor NR2F6.

NR2F6 is considered an orphan nuclear receptor since its endogenous ligand has yet to be identified. Recently, NR2F6 has emerged as a novel cancer therapeutic target. NR2F6 has been demonstrated to be upregulated or overexpressed in several cancers. Importantly, Nr2f6-/- mice spontaneously reject tumors and develop host-protective immunological memory, a consequence of NR2F6 acting as an immune checkpoint in effector T cells. Collectively, these data suggest that modulation of NR2F6 activity may have important clinical applications in the fight against cancer. The nuclear receptor superfamily of ligand-regulated transcription factors has proven to be an excellent source of targets for therapeutic intervention of a broad range of diseases. Approximately 15% of FDA approved drugs target NRs, demonstrating their clinical efficacy. To identify small molecule regulators of NR2F6 activity, with the overall goal of immuno-oncology, we developed and initiated a high-throughput cell-based assay that specifically measures the transcriptional activity of NR2F6. We completed automated screening of approximately 666,000 compounds and identified 5,008 initial hits. Further screening efforts, including counterscreening assays, confirmed 128 of these hits, most of which had IC50s of equal to or less than 5μM potencies. Here, we report, for the first time, the identification of several small molecule compounds to the orphan nuclear receptor, NR2F6.