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Aberrant splicing is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. The oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors have been demonstrated in multiple studies. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for the treatment of insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore technology is an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with the potential to translate into therapeutic candidates for TNBC. Interestingly, one of our leading AS targets is an exon skipping isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival, without showing differences in gene expression between all the breast cancer subtypes and healthy patients studied. In addition, SpliceCore was used to predict and design a set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. The skipping isoform plays a critical role in tumor progression via a TGFβ-dependent mechanism as demonstrated by detailed isoform switching studies using SSO-0205. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation modulated TGFβ pathway related protein levels and cellular localization and reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. In summary, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Our results provide experimental proof of concept that demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent therapeutic development. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Moreover, SpliceCore can be applied to multiple other indications opening a new avenue for therapeutic development in cancer. Citation Format: Miguel A Manzanares, Priyanka Dhingra, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. Novel therapeutic target for triple negative breast cancer uncovered by SpliceCore® an innovative platform that identifies disease-specific alternative splicing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-11.

This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of novel and verifiable splice-switching oligonucleotide (SSO) targetsin-silico. SSOs are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). To leverage the potential of AS research for therapeutic development, we created SpliceLearn™, an AI/ML algorithm for the identification of modulatory SSO binding sites on pre-mRNA. SpliceLearn also predicts the identity of specific splicing factors whose binding to pre-mRNA is blocked by SSOs, adding considerable transparency to AI/ML-driven drug discovery and informing biological insights useful in further validation steps. Here we predictedNEDD4Lexon 13 (NEDD4Le13) as a novel target in triple negative breast cancer (TNBC) and computationally designed an SSO to modulateNEDD4Le13. TargetingNEDD4Le13with this SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFβ pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data. SpliceLearn is part of the SpliceCore® platform, an AI/ML predictive ensemble for AS-based drug target discovery.

RNA splicing is a central step in the regulation of gene expression, leading to the accurate assembly of viable mRNA isoforms. Errors in the splicing process are common in cancer and lead to the production of tumor-specific mRNAs that often encode for neoantigen peptides. Such neoantigens cannot be discovered using conventional DNA-based tools; they require specialized software for alternative splicing analysis using RNA-seq data. Envisagenics has developed SpliceIOTM, a proprietary software platform for splicing-derived neoantigen discovery. SpliceIO uses RNA-seq data to uncover extracellular neoantigens encoded by transmembrane proteins or presented to immune cells through the MHC complex. Using SpliceIO, we have analyzed roughly 8,000 RNA-seq samples from 4 different cancers and 14 normal tissue types. Each dataset first undergoes an unsupervised clustering analysis using SpliceSliceTM to stratify patients into subpopulations based on similar aberrations in splicing machinery. We then leverage SpliceIO to identify splicing-derived, tumor-specific neoantigens enriched in these cohorts. SpliceIO incorporates features, such as protein localization, MHC binding affinity, epitope accessibility and cancer relevance of the gene to help select tumor specific targets with the highest therapeutic potential. Here, we summarize the discovery and validation of novel splicing-derived neoantigens identified using SpliceIO. To validate neoantigen candidates, we performed isoform expression quantification at the RNA and protein levels using PCR, RNA-FISH, western blot and mass spectrometry. Our experiments confirmed several neoantigen candidates encoded by tumor-specific RNA isoforms, that are translated and localized to the cellular membrane. Interestingly, one of the candidates is an endoplasmic reticulum protein that exhibits cassette exon skipping in approximately 24% of TNBC tumors. Fluorescence microscopy experiments demonstrate that the TNBC-specific isoform of the protein is localized to plasma membrane but not the ER, suggesting that alternative splicing can alter protein localization. Together, our results demonstrate the potential of RNA splicing analysis for the discovery of novel immunotherapeutic targets. Citation Format: Hasan Ekrem Zumrut, Kendall Anderson, Miguel Manzanares, Shaleigh Smith, Sakshi Gera, Nayab Habib, Sanjana Shah, Devon Trahan, Martin Akerman, Gayatri Arun. Computational discovery and experimental validation of splicing-derived neoantigens [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 448.

Sachet water is one of the primary sources of drinking water in rapidly growing countries. A study to assess the microbiological quality of sachet water in 21 different brands was conducted in Ghana. Culturable total coliform was positive in 87% of the samples collected, where Escherichia coli colonies were absent. The analysis of quantitative polymerase chain reaction results indicated the presence of E. coli genes in 44.6% of the samples, with the highest concentration up to 3,166 CCE/100 ml. Microbial source tracking analyses showed that the source of E. coli genes did not originate from sewage contamination because the human-associated HF183 marker was not detected. Of the 175 samples tested, 71% did not mention any water treatment before filling the packages. These results suggest non-human sources of contamination, such as biofilm formation in the pipelines used to fill these packages due to poor disinfection. Our study shows an urgent need for increased regulation and standardized manufacturing of sachet water to ensure safe drinking water.

Accumulating evidence highlights aberrant splicing as a disease-driving event in cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have described the tumor-promoting activity of specific aberrant cis-splicing variants and misregulation of trans-acting splicing factors in patients. Hence, the identification of aberrant splicing patterns has the potential to translate into actionable biomarkers and novel therapeutic targets, particularly for treatment insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore is an innovative cloud-based platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor-specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNA-seq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS derived therapeutic targets with the potential to translate into therapeutic candidates for TNBC. Here, we report a novel, alternatively spliced isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival. This cytoplasmic protein was previously described as a regulator of the TGFβ pathway; therefore, we have validated the isoform switching action in a TGFβ dependent tumor progression model and demonstrated the activity of Splice Switching Oligos (SSO) to modulate its effect. We observed that this AS was correlated to a differential response on subcellular localization for phSmad2/3, Smad2/3 and TGFβRI. Pretreatment of the TNBC cells with SSO before TGFβ pathway activation modulates its proliferation response by affecting gene expression of p21, c-Myc and Smad7, leading to a decrease of cells in the G2 mitotic phase of the cell cycle and a loss of cell viability after the SSO treatment. More importantly, migratory response induced by TGFβ in TNBC cells was significantly inhibited by SSOs. Pre-treatment with SSO-0205 before TGFβ activation downregulated gene expression of Angiopoietin-like 4, Integrins α5 and β3, TGFβ associated migration markers, which was followed by a 55% decrease in cell migration. Our state-of-the-art ML technology, SpliceCore, has proven its ability to uncover novel disease-specific AS targets and to design splice correcting oligonucleotides for subsequent therapeutic development. Our data provides experimental proof of concept that has uncovered a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by misregulation of the TGFβ pathway. Moreover, SSOs predicted by SpliceCore were able to correct the aberrant splicing downstream effect, thereby uncovering a novel alternative splicing targeted approach for treating TNBC patients. Citation Format: Miguel A. Manzanares, Alyssa Casill, Kendall Anderson, Priyanka Dhingra, Vanessa Frederick, Adam Geier, Martin Akerman, Gayatri Arun. SpliceCore® a novel ML tool for identifying disease-specific alternative splicing uncovered a promising therapeutic target for triple negative breast cancer [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 1873.

Ozonation is widely used in high-income countries for water disinfection in centralized treatment facilities. New microplasma technology has reduced the energy requirements for ozone generation dramatically, such that a 15-watt solar panel is sufficient to produce small quantities of ozone. This technology has not been used previously for point-of-use drinking water treatment. We conducted a series of assessments of this technology, both in the laboratory and in homes of residents of a village in western Kenya, to estimate system efficacy and to determine if the solar-powered point-of-use water ozonation system appears safe and acceptable to end-users. In the laboratory, two hours of point-of-use ozonation reduced E. coli in 120 L of wastewater by a mean (standard deviation) of 2.3 (0.84) log-orders of magnitude and F+ coliphage by 1.54 (0.72). Based on laboratory efficacy, 10 families in Western Kenya used the system to treat 20 L of household stored water for two hours on a daily basis for eight weeks. Household stored water E. coli concentrations of &gt, 1000 most probable number (MPN)/100 mL were reduced by 1.56 (0.96) log removal value (LRV). No participants experienced symptoms of respiratory or mucous membrane irritation. Focus group research indicated that families who used the system for eight weeks had very favorable perceptions of the system, in part because it allowed them to charge mobile phones. Drinking water ozonation using microplasma technology may be a sustainable point-of-use treatment method, although system optimization and evaluations in other settings would be needed.

Most coastal freshwater ecosystems in the United States have semi-tidal movements during the day. Routine monitoring of these environments is conducted once during the day when tides can be at either ebb or flood conditions, causing a variability in bacterial concentrations and misinterpretation of the illness risk associated with human activities. The occurrence and levels of enterococci (enterococci 23S rDNA [Ent23S]) and human- (HF183) and avian- (GFD) associated microbial source tracking (MST) markers were investigated using quantitative polymerase chain reaction (qPCR) along with detection of culturable enterococci and environmental parameters. Samples were collected during flood and ebb tide conditions (May-September) from a tidal river used for recreational activities. Culturable enterococci [(420) = 2.093, = 0.040] and Ent23S [(420) = 2.243, = 0.028] controlled for tide type were significantly different; higher enterococci concentrations were detected during the flood tide. Among all samples, 6% were positive for HF183, and GFD was positively correlated with Ent23S ( = 0.92, = 0.029) and conductivity ( = 0.93, = 0.023) during flood tide. Unlike the general assumption that ebb tide flow in a river would likely carry runoff from the land, the microbial contaminants in this case were transported from upstream via ocean water to the river during the flood tide. These results suggest that hydrology and land use patterns must be considered in sampling design when conducting future microbial water quality monitoring programs to better characterize recreational water safety in tidal rivers.

Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for treatment insensitive cancers such as TNBC. Envisagenics has developed SpliceCore, an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with a potential to translate into therapeutic candidates for TNBC. Here, we will discuss one of our most promising AS targets; it is present in 60.5% of TNBC patients and correlates with poor overall survival. Using SpliceCore, we predicted and designed an optimal set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. Detailed mechanism of action studies of isoform switching by SSO-0205 have demonstrated the critical role of the isoform in a TGFβ dependent tumor progression mechanism. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. Together, we provide experimental proof of concept to demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent correction and therapeutic development. Using this approach, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Citation Format: Miguel A. Manzanares, Dhingra Priyanka, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P170.