Cancer-specific Gene Research Articles

Abstract A063: CancerTools.org: Research Tools Supporting Breast Cancer Studies

Abstract CancerTools.org is the latest, non-profit cancer-focused biorepository. Driven by a mission to accelerate cancer research discoveries, the platform allows scientists to deposit and access tangible laboratory research materials. Till date, researchers from over 175 academic institutes globally have already deposited their tools, generating a cutting-edge portfolio of over 5,000 reagents, including a range of cell-based models and antibodies. Breast cancer is one of the most frequent malignancies in women worldwide. A comprehensive collection of tools has been curated at CancerTools.org to support this important area of research. Cell-based models include the RMAM007-3 breast cancer organoid which recapitulates in vivo tumour characteristics and is suitable for disease modelling, 3D imaging, and anticancer drug discovery. Cell lines such as human anti-oestrogen resistant lines developed from MCF-7 and T47D cells, demonstrate resistance to hormone-dependent treatments and are useful for studying therapy resistance mechanisms and biomarker development for breast cancer detection. NCI-HPN-F murine cell lines with recurrent chromosomal aneuploidies, have enabled investigation and identification of tumorigenesis related cancer-specific genes and signalling pathways. Further models include gene-edited cell lines to explore BRCA1 hypermethylation pathways; reporter breast cancer cell lines to assess apoptosis, necrosis, and autophagy; and immortalised human breast epithelial cell lines to study differentiation and phenotypic transformation. To provide a physiologically relevant cell culture environment, PlasmaxTM, a defined media that mirrors the concentration of over 50 components to human plasma, is also part of the repertoire. It has been used to culture triple-negative breast cancer cell lines to recapitulate the tumour metabolic environment. Antibodies to study breast cancer biology, including tumour suppressors, angiogenesis and metastasis, form part of the portfolio. Antibodies targeting tumour suppressors include RB1, which is often lost by mutation, deletion, transcriptional silencing and hyper-phosphorylation in malignancies- including breast cancer. Deletion and rearrangement of RB1 is estimated in ~20-25% of breast cancer cell lines. Tools identifying specific RB1 phosphorylation sites, include Anti-Phospho-Rb for Ser608 and Thr356. Additional insights into breast cancer prognosis are offered by Anti-Maspin [16F7] which recognises wildtype and denaturated forms of the protein. Maspin is an inhibitor of serine proteases with tumour suppressor activity in breast cancer. Its nuclear staining associates with favourable outcomes in contrast to cytoplasmic staining. MUC1 is also aberrantly overexpressed in 90% of human breast cancer, thus, representing a promising marker for breast cancer diagnosis and prognosis. Further research support is offered by an extensive range of antibodies for MUC1’s different isoforms. Visit our stand to explore more about depositing and accessing tools and technologies that support breast cancer research. Citation Format: Vera Moiseeva, Sara Omenetti, Abby Henney, Katie Valencia. CancerTools.org: Research Tools Supporting Breast Cancer Studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A063.

Towards targeting transposable elements for cancer therapy.

Transposable elements (TEs) represent almost half of the human genome. Historically deemed 'junk DNA', recent technological advancements have stimulated a wave of research into the functional impact of TEs on gene-regulatory networks in evolution and development, as well as in diseases including cancer. The genetic and epigenetic evolution of cancer involves the exploitation of TEs, whereby TEs contribute directly to cancer-specific gene activities. This Review provides a perspective on the role of TEs in cancer as being a 'double-edged sword', both promoting cancer evolution and representing a vulnerability that could be exploited in cancer therapy. We discuss how TEs affect transcriptome regulation and other cellular processes in cancer. We highlight the potential of TEs as therapeutic targets for cancer. We also summarize technical hurdles in the characterization of TEs with genomic assays. Last, we outline open questions and exciting future research avenues.

Network pharmacology: an efficient but underutilized approach in oral, head and neck cancer therapy-a review.

The application of network pharmacology (NP) has advanced our understanding of the complex molecular mechanisms underlying diseases, including neck, head, and oral cancers, as well as thyroid carcinoma. This review aimed to explore the therapeutic potential of natural network pharmacology using compounds and traditional Chinese medicines for combating these malignancies. NP serves as a pivotal tool that provides a comprehensive view of the interactions among compounds, genes, and diseases, thereby contributing to the advancement of disease treatment and management. In parallel, this review discusses the significance of publicly accessible databases in the identification of oral, head, and neck cancer-specific genes. These databases, including those for head and neck oral cancer, head and neck cancer, oral cancer, and genomic variants of oral cancer, offer valuable insights into the genes, miRNAs, drugs, and genetic variations associated with these cancers. They serve as indispensable resources for researchers, clinicians, and drug developers, contributing to the pursuit of precision medicine and improved treatment of these challenging malignancies. In summary, advancements in NP could improve the globalization and modernization of traditional medicines and prognostic targets as well as aid in the development of innovative drugs. Furthermore, this review will be an eye-opener for researchers working on drug development from traditional medicines by applying NP approaches.

Super-enhancer-driven expression of BAHCC1 promotes melanoma cell proliferation and genome stability

Super-enhancers (SEs) are stretches of enhancers ensuring a high level of expression of key genes associated with cell function. The identification of cancer-specific SE-driven genes is a powerful means for the development of innovative therapeutic strategies. Here, we identify a MITF/SOX10/TFIIH-dependent SE promoting the expression of BAHCC1 in a broad panel of melanoma cells. BAHCC1 is highly expressed in metastatic melanoma and is required for tumor engraftment, growth, and dissemination. Integrative genomics analyses reveal that BAHCC1 is a transcriptional regulator controlling expression of E2F/KLF-dependent cell-cycle and DNA-repair genes. BAHCC1 associates with BRG1-containing remodeling complexes at the promoters of these genes. BAHCC1 silencing leads to decreased cell proliferation and delayed DNA repair. Consequently, BAHCC1 deficiency cooperates with PARP inhibition to induce melanoma cell death. Our study identifies BAHCC1 as an SE-driven gene expressed in melanoma and demonstrates how its inhibition can be exploited as a therapeutic target.

HHCDB: a database of human heterochromatin regions.

Heterochromatin plays essential roles in eukaryotic genomes, such as regulating genes, maintaining genome integrityand silencing repetitive DNA elements. Identifying genome-wide heterochromatin regions is crucial for studying transcriptional regulation. We propose the Human Heterochromatin Chromatin Database (HHCDB) for archiving heterochromatin regions defined by specific or combined histone modifications (H3K27me3, H3K9me2, H3K9me3) according to a unified pipeline. 42839743 heterochromatin regions were identified from 578 samples derived from 241 cell-types/cell lines and 92 tissue types. Genomic information is provided in HHCDB, including chromatin location, gene structure, transcripts, distance from transcription start site, neighboring genes, CpG islands, transposable elements, 3D genomic structureand functional annotations. Furthermore, transcriptome data from 73 single cells were analyzed and integrated to explore cell type-specific heterochromatin-related genes. HHCDB affords rich visualization through the UCSC Genome Browser and our self-developed tools. We have also developed a specialized online analysis platform to mine differential heterochromatin regions in cancers. We performed several analyses to explore the function of cancer-specific heterochromatin-related genes, including clinical feature analysis, immune cell infiltration analysis and the construction of drug-target networks. HHCDB is a valuable resource for studying epigenetic regulation, 3D genomicsand heterochromatin regulation in development and disease. HHCDB is freely accessible at http://hhcdb.edbc.org/.

Comprehensive genomic profiling testing in Japanese castration-resistant prostate cancer patients: results of a single-center retrospective cohort study.

Comprehensive genomic profiling testing using a hybrid-capture next-generation sequencing is commonly used in clinical practice to employ precision medicine in cancer treatment worldwide. In this study, we aimed to analyze the profiles obtained using comprehensive genomic profiling testing that was performed in Japanese castration-resistant prostate cancer patients and to discuss the genetic findings in a real-world setting. A total of 60 cases and 57 castration-resistant prostate cancer patients underwent comprehensive genomic profiling testing between 1 January 2021 and 31 December 2022. Four types of comprehensive genomic profiling testing were selected, and clinically significant cancer-specific gene alterations were identified. The median age of patients was 74years, and the median prostate-specific antigen value at the time of submission was 18.6ng/ml. Fifty-seven (95%) of 60 cases were metastatic castration-resistant prostate cancers, and 3 cases (5%) were non-metastatic. Among all genetic alterations, androgen-receptor alteration was the most frequently detected in 17 cases (28.3%), followed by 15 cases of TP53 (25.0%), 14 cases of CDK12 (23.3%), 10 cases of phosphatase and tensin homolog (16.7%) and 9 cases of ATM (15.0%) mutations. A total of 13 patients (21.7%) received systemic therapy according to the comprehensive genomic profiling testing results. Overall, the survival rate was significantly greater in the group treated through systemic therapy based on comprehensive genomic profiling testing compared with the group without new therapeutic treatment (P=0.041). Comprehensive genomic profiling testing is recommended in castration-resistant prostate cancer patients identified as resistant to standard therapy as this can provide a new therapeutic option.

Genetic and Functional Modifications Associated with Ovarian Cancer Cell Aggregation and Limited Culture Conditions.

The aggregation of cancer cells provides a survival signal for disseminating cancer cells; however, the underlying molecular mechanisms have yet to be elucidated. Using qPCR gene arrays, this study investigated the changes in cancer-specific genes as well as genes regulating mitochondrial quality control, metabolism, and oxidative stress in response to aggregation and hypoxia in our progressive ovarian cancer models representing slow- and fast-developing ovarian cancer. Aggregation increased the expression of anti-apoptotic, stemness, epithelial-mesenchymal transition (EMT), angiogenic, mitophagic, and reactive oxygen species (ROS) scavenging genes and functions, and decreased proliferation, apoptosis, metabolism, and mitochondrial content genes and functions. The incorporation of stromal vascular cells (SVF) from obese mice into the spheroids increased DNA repair and telomere regulatory genes that may represent a link between obesity and ovarian cancer risk. While glucose had no effect, glutamine was essential for aggregation and supported proliferation of the spheroid. In contrast, low glucose and hypoxic culture conditions delayed adhesion and outgrowth capacity of the spheroids independent of their phenotype, decreased mitochondrial mass and polarity, and induced a shift of mitochondrial dynamics towards mitophagy. However, these conditions did not reduce the appearance of polarized mitochondria at adhesion sites, suggesting that adhesion signals that either reversed mitochondrial fragmentation or induced mitobiogenesis can override the impact of low glucose and oxygen levels. Thus, the plasticity of the spheroids' phenotype supports viability during dissemination, allows for the adaptation to changing conditions such as oxygen and nutrient availability. This may be critical for the development of an aggressive cancer phenotype and, therefore, could represent druggable targets for clinical interventions.

Systemic Delivery of Magnetogene Nanoparticle Vector for Gene Expression in Hypoxic Tumors.

Cancer is a disease that causes millions of deaths per year worldwide because conventional treatments have disadvantages such as unspecific tumor selectivity and unwanted toxicity. Most human solid tumors present hypoxic microenvironments and this promotes multidrug resistance. In this study, we present "Magnetogene nanoparticle vector" which takes advantage of the hypoxic microenvironment of solid tumors to increase selective gene expression in tumor cells and reduce unwanted toxicity in healthy cells; this vector was guided by a magnet to the tumor tissue. Magnetic nanoparticles (MNPs), chitosan (CS), and the pHRE-Luc plasmid with a hypoxia-inducible promoter were used to synthesize the vector called "Magnetogene nanoparticles" by ionic gelation. The hypoxic functionality of Magnetogene vector nanoparticles was confirmed in the B16F10 cell line by measuring the expression of the luciferase reporter gene under hypoxic and normoxic conditions. Also, the efficiency of the Magnetogene vector was confirmed in vivo. Magnetogene was administered by intravenous injection (IV) in the tail vein and directed through an external magnetic field at the site of tumor growth in C57Bl/6 mice. A Magnetogene vector with a size of 50 to 70 nm was directed and retained at the tumor area and gene expression was higher at the tumor site than in the others tissues, confirming the selectivity of this vector towards hypoxic tumor areas. This nanosystem, that we called the "Magnetogene vector" for systemic delivery and specific gene expression in hypoxic tumors controlled by an external magnetic designed to target hypoxic regions of tumors, can be used for cancer-specific gene therapies.

Comprehensive information-based differential gene regulatory networks analysis (CIdrgn): Application to gastric cancer and chemotherapy-responsive gene network identification.

Biological condition-responsive gene network analysis has attracted considerable research attention because of its ability to identify pathways or gene modules involved in the underlying mechanisms of diseases. Although many condition-specific gene network identification methods have been developed, they are based on partial or incomplete gene regulatory network information, with most studies only considering the differential expression levels or correlations among genes. However, a single gene-based analysis cannot effectively identify the molecular interactions involved in the mechanisms underlying diseases, which reflect perturbations in specific molecular network functions rather than disorders of a single gene. To comprehensively identify differentially regulated gene networks, we propose a novel computational strategy called comprehensive analysis of differential gene regulatory networks (CIdrgn). Our strategy incorporates comprehensive information on the networks between genes, including the expression levels, edge structures and regulatory effects, to measure the dissimilarity among networks. We extended the proposed CIdrgn to cell line characteristic-specific gene network analysis. Monte Carlo simulations showed the effectiveness of CIdrgn for identifying differentially regulated gene networks with different network structures and scales. Moreover, condition-responsive network identification in cell line characteristic-specific gene network analyses was verified. We applied CIdrgn to identify gastric cancer and itsf chemotherapy (capecitabine and oxaliplatin) -responsive network based on the Cancer Dependency Map. The CXC family of chemokines and cadherin gene family networks were identified as gastric cancer-specific gene regulatory networks, which was verified through a literature survey. The networks of the olfactory receptor family with the ASCL1/FOS family were identified as capecitabine- and oxaliplatin sensitive -specific gene networks. We expect that the proposed CIdrgn method will be a useful tool for identifying crucial molecular interactions involved in the specific biological conditions of cancer cell lines, such as the cancer stage or acquired anticancer drug resistance.

Expression profiling of luminal B breast tumor in Indian women.

In this study, we aimed at profiling of luminal B breast cancer specific gene expression pattern in Indian women using mRNA-seq and validation based on TCGA expression data. RNA isolated from luminal B tumor and adjacent normal tissues was used for library construction and sequencing. Reference-based assemblies of these reads were used for differential gene expression analysis using DeSeq2. The DEGs were evaluated using TCGA expression data. Kaplan-Meier survival method was used to evaluate association between genes showing luminal B specific differential expression pattern and breast cancer prognosis and statistical significance was assessed using log-rank test. Alternate splicing analysis was done using rmats. Differential expression analysis identified 2371 differentially expressed genes (DEGs) in luminal B breast tumors in comparison with adjacent normal tissues of Indian Women. Of them, 1692 DEGs were validated using TCGA luminal B paired samples. Integration of this data with the DEGs obtained by comparative analysis of unpaired luminal B with luminal A unpaired samples from TCGA resulted in 291 DEGs showing luminal B specific expression pattern. Further, 26 genes of prognostic value were identified. Differential splicing analysis between luminal B tumors and adjacent normal tissues in our cohort led to the identification of 687 genes showing significant differential alternate splicing events. This study profiled gene expression pattern of luminal B tumors of Indian women and identified 26 key genes of prognostic value for luminal B breast cancer. This study also profiled differential alternate splicing and identified important alternate splicing events in luminal B breast cancer.

In Vitro Prostate Cancer Treatment via CRISPR-Cas9 Gene Editing Facilitated by Polyethyleneimine-Derived Graphene Quantum Dots.

CRISPR-Cas9 is a programmable gene editing tool with a promising potential for cancer gene therapy. This therapeutic function is enabled in the present work via the non-covalent delivery of CRISPR ribonucleic protein (RNP) by cationic glucosamine/PEI-derived graphene quantum dots (PEI-GQD) that aid in overcoming physiological barriers and tracking genes of interest. PEI-GQD/RNP complex targeting the TP53 mutation overexpressed in ~50% of cancers successfully produces its double-stranded breaks in solution and in PC3 prostate cancer cells. Restoring this cancer "suicide" gene can promote cellular repair pathways and lead to cancer cell apoptosis. Its repair to the healthy form performed by simultaneous PEI-GQD delivery of CRISPR RNP and a gene repair template leads to a successful therapeutic outcome: 40% apoptotic cancer cell death, while having no effect on non-cancerous HeK293 cells. The translocation of PEI-GQD/RNP complex into PC3 cell cytoplasm is tracked via GQD intrinsic fluorescence, while EGFP-tagged RNP is detected in the cell nucleus, showing the successful detachment of the gene editing tool upon internalization. Using GQDs as non-viral delivery and imaging agents for CRISPR-Cas9 RNP sets the stage for image-guided cancer-specific gene therapy.

Multi-Omics Data Analysis Identifies Prognostic Biomarkers across Cancers.

Combining omics data from different layers using integrative methods provides a better understanding of the biology of a complex disease such as cancer. The discovery of biomarkers related to cancer development or prognosis helps to find more effective treatment options. This study integrates multi-omics data of different cancer types with a network-based approach to explore common gene modules among different tumors by running community detection methods on the integrated network. The common modules were evaluated by several biological metrics adapted to cancer. Then, a new prognostic scoring method was developed by weighting mRNA expression, methylation, and mutation status of genes. The survival analysis pointed out statistically significant results for GNG11, CBX2, CDKN3, ARHGEF10, CLN8, SEC61G and PTDSS1 genes. The literature search reveals that the identified biomarkers are associated with the same or different types of cancers. Our method does not only identify known cancer-specific biomarker genes, but also proposes new potential biomarkers. Thus, this study provides a rationale for identifying new gene targets and expanding treatment options across cancer types.

Wnt Signaling Stimulates Cooperation between GREB1 and HNF4α to Promote Proliferation in Hepatocellular Carcinoma.

GREB1 is a liver cancer-specific Wnt signaling target gene that induces an oncogenic shift of HNF4α, a putative tumor suppressor, and may represent a therapeutic target in Wnt-activated hepatocellular carcinoma.

Abstract A055: NSD2 is a requisite and targetable subunit of the AR/FOXA1 neo-enhanceosome complex in prostate cancer cells

Abstract The androgen receptor (AR) is a ligand-responsive transcription factor (TF) that binds as a homodimer at FOXA1-pioneered enhancer elements containing a palindromic DNA motif. Prostate cancer (PCa) is highly dependent on the AR enhanceosome complex, and in castration-resistant disease, this dependency is reinforced through alterations in the AR pathway. This centrally involves extensive rewiring of the AR cistrome to gain de novo binding at enhancer sites (aka neo-enhancers) that activate hyperproliferative and metastatic gene programs. However, requisite subunits of the AR neo-enhanceosome and the genes it uniquely activates remain uncharacterized. Here, we CRISPR-engineered an endogenous AR reporter system by fusing the mCherry coding sequence in-frame with the KLK3/PSA gene and conducted a CRISPR knock-out screen to identify druggable chromatin/epigenetic cofactors of AR. Using a sgRNA library covering >200 druggable transcriptional coregulators, ranked alongside BRD4 and TRIM24/28, we identified NSD2 as a novel AR coactivator. NSD2 is an H3K36 di-methyltransferase that activates gene expression through antagonism of the PRC2/EZH2 complex. In PCa cells, genetic inhibition of NSD2 function significantly attenuated the expression of AR targets without affecting the AR protein level, suggesting that NSD2 regulates either AR’s chromatin assembly or trans-activational competence. Cistromic profiling of AR (ChIPseq) in NSD2-null PCa cells showed complete loss of binding at >40,000 genomic sites (~65% of the cistrome), with HOMER motif analyses of NSD2-dependent AR sites showing a higher recurrence of hexameric 5′-AGAACA-3′ AR half-site juxtaposed to the FOXA1, ETS, or HOXB13 motifs. In contrast, NSD2-independent AR sites, a large fraction of which show increased binding in NSD2-null cells, housed the canonical 15-bp palindromic AR motif with two invertedly-oriented half-sites separately recognized by each half of the AR homodimer. Notably, analyses of primary AR cistromes from human tissues revealed AR loading at half-motifs to be 20-30-fold enriched in the tumor-specific AR enhancer circuitry. In phenotypic assays, we found NSD2-deficient PCa cells to lose cancer hallmarks such as colony formation, invasion, and hyper-proliferation. We further developed a potent NSD2 PROTAC, LLC0150, which showed preferential cytotoxicity in AR/FOXA1+ PCa relative to the AR-negative disease, and other cancer and normal cell lines from 22 distinct lineages. In a pan-cancer screen comprising over 100 cell lines, treatment with LLC0150 triggered apoptotic death in AR/FOXA1+ malignancies and NSD2-mutant acute lymphocytic leukemia, and multiple myeloma. Altogether, we identify NSD2 as a novel AR neo-cofactor that assists oncogenic TFs in loading the AR enhanceosome at degenerate, low-affinity AR elements, thereby wiring its cancer-specific gene programs. We develop a novel NSD2 PROTAC that selectively kills AR/FOXA1-driven prostate and NSD2-altered tumors in preclinical models, positioning NSD2 therapeutics for safety and efficacy evaluation in human clinical trials. Citation Format: Abhijit Parolia, Brijesh K. Verma, Sanjana Eyunni, Sweta Aras, Sandra E. Carson, Eleanor Young, Chandan K. Das, James M. George, Reyaz ur Rasool, Prathibha Gajjala, Mohammed Alhusayan, Erick Mitchell-Velasquez, Lanbo Xiao, Jie Luo, Mustapha Jaber, Xuhong Cao, Fengyun Su, Rui Wang, Lianchao Liu, Zhen Wang, Ke Ding, Arul M. Chinnaiyan, Irfan Asangani. NSD2 is a requisite and targetable subunit of the AR/FOXA1 neo-enhanceosome complex in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A055.

Cancer Therapeutic siRNA Delivery and Imaging by Nitrogen- and Neodymium-Doped Graphene Quantum Dots.

While small interfering RNA (siRNA) technology has become a powerful tool that can enable cancer-specific gene therapy, its translation to the clinic is still hampered by the inability of the genes alone to cell transfection, poor siRNA stability in blood, and the lack of delivery tracking capabilities. Recently, graphene quantum dots (GQDs) have emerged as a novel platform allowing targeted drug delivery and fluorescence image tracking in visible and near-infrared regions. These capabilities can aid in overcoming primary obstacles to siRNA therapeutics. Here, for the first time, we utilize biocompatible nitrogen- and neodymium-doped graphene quantum dots (NGQDs and Nd-NGQDs, respectively) for the delivery of Kirsten rat sarcoma virus (KRAS) and epidermal growth factor receptor (EGFR) siRNA effective against a variety of cancer types. GQDs loaded with siRNA noncovalently facilitate successful siRNA transfection into HeLa cells, confirmed by confocal fluorescence microscopy at biocompatible GQD concentrations of 375 μg/mL. While the GQD platform provides visible fluorescence tracking, Nd doping enables deeper-tissue near-infrared fluorescence imaging suitable for both in vitro and in vivo applications. The therapeutic efficacy of the GQD/siRNA complex is verified by successful protein knockdown in HeLa cells at nanomolar siEGFR and siKRAS concentrations. A range of GQD/siRNA loading ratios and payloads are tested to ultimately provide substantial inhibition of protein expression down to 31-45%, comparable with conventional Lipofectamine-mediated delivery. This demonstrates the promising potential of GQDs for the nontoxic delivery of siRNA and genes in general, complemented by multiwavelength image tracking.

Molecular targeted therapy, advanced treatment for cancers of the head-and-neck region: A systematic review.

The foundation of oncology treatment as a single modality approach as well as the "multimodality" concept has been studied by statistical evaluation pre, during, and posttreatment to rule out their efficacy, expected prognosis, toxicity reactions, and overall survival for the patient. Such studies have also provided an appreciable amount of data for future custom utility. "Targeted therapy" is a cancer treatment that uses drugs but is different from traditional chemotherapy. It works by targeting cancer-specific genes, proteins, or the tissue environment that contributes to cancer growth and survival. Researchers are developing drugs that target specific molecular changes. The drugs can block or turn off signals that tell cancer cells to grow and divide, keep cells from living longer than usual, and destroy the cancer cells. The aim of the study is to carry out a systematic review of clinical trials of molecular targeted therapy in the treatment of cancer. The objective of the study is to evaluate the efficacy of molecular targeted therapy in the treatment of head-and-neck cancers. A group of keywords was preselected to search for scientific articles on a web-based database of PubMed. Only completed randomized controlled trials published in the past 5 years in the English language were included with open access. All the selected articles were subjected to the Cochrane bias tool and PRISMA guidelines to extract results. Among 4 studies specifying the progression-free survival (PFS) for comparing the groups treated either using targeted therapy or other modality/placebo, 50% of studies show a slight increase in PFS in the group treated with TT and other 50% show PFS increase in the non-TT group. Thus, insufficient evidence is furnished to provide a statement and acknowledged the expectancy of a disease-free period with or without the use of TT in the treatment of head-and-neck cancer. Considering very little information on enhanced effect and presence of evidence supporting an increased risk of adverse events, the addition of TT to treatment is a question to the dilemma. A systematic review intends advantageous in providing foresight for oncologists concerning patient assessment and evaluation to defend inclination proceeding toward the treatment defined.

Abstract LB234: Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800

Abstract HSF1 is a stress-inducible transcription factor that regulates the eukaryotic heat shock response (HSR). HSF1 activation induces the expression of multiple proteins needed for cellular recovery from stress. HSF1 also plays a key role in tumorigenesis and regulates the expression of a cancer-specific gene signature which is unique to malignant cells and distinct from that activated during the HSR. We discovered NXP800, the first-in-class orally active HSF1 pathway inhibitor which is now undergoing Phase 1 clinical trial, based on a phenotypic pathway screen. Here we employed various techniques to investigate the mechanism of action of NXP800. Using RNAseq in a panel of human carcinoma cell lines, we identified overlapping gene expression changes in response to NXP800. These included genes regulated by HSF1 and interestingly also genes associated with activation of the integrated stress response (ISR). In contrast, we found no evidence for activation of the unfolded protein response. Consistent with the observed NXP800-induced phosphorylation of eIF2α which is a critical regulator of the ISR, NXP800 increased the protein expression of downstream ISR markers ATF4, CHOP and CHAC1, both in human tumor cells in vitro and in human tumor xenograft models in vivo. Induction of the ISR is controlled by four stress-activated protein kinases (PKs) that phosphorylate eIF2α. To further explore these kinases in the mechanism of action of NXP800 we used genetic knockdown by siRNA and inhibition by small-molecule tool compounds. Silencing each of the ISR-regulatory PKs revealed that GCN2 was required for ISR activation by NXP800. This was confirmed using two GCN2 inhibitors from different chemical series. Global phospho-proteome analysis showed that altered protein phosphorylation following NXP800 exposure was reversed upon co-treatment with a GCN2 inhibitor. We also demonstrated that activation of the ISR caused inhibition of HSF1 activation when stimulated with an HSF1 activator, thus confirming the link between ISR induction and inhibition of HSF1 activation. Activation of GCN2 and the ISR can occur in response to a variety of stimuli including amino acid deprivation. However, we did not detect a difference in the uptake of amino acids following exposure to NXP800, indicating that NXP800 does not directly impair amino acid uptake. Using an siRNA approach to determine if activation of the ISR components was contributing to growth inhibition following NXP800 exposure, we found that blocking the induction of ATF4 reduced the response of NXP800-sensitive SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. In summary, NXP800 acts on cancer cells to induce activation of the ISR pathway via GCN2, which then leads to inhibition of HSF1 activation. Further studies are underway to determine the precise molecular target of NXP800 and the mechanism of HSF1 pathway inhibition. Citation Format: Marissa V. Powers, Swee Y. Sharp, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Robert te Poele, Florence Raynaud, Suzanne Eccles, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB234.

Abstract 5368: AI-enabled prediction of lung cancer specific hot spot gene alterations from histology images

Abstract Background: The clinical intervention relies on molecular testing of primary biomarkers. We developed an AI enabled platform to predict NCCN guidelines recommended hot-spot genomic alterations from the image analysis using H&E slide. Methods: AI engine was trained using clinically observed hot spot genomic alterations present in lung cancer patients as a ground truth based on histological FFPE whole-slide images. The H & E stained histological images of the tumor having matching pathogenic mutations were downloaded from the TCGA database for the training and testing purpose. The program extracted histopathological features, followed by pattern mapping to predict genomics status based on the results of NGS testing from 480 samples. ResNet AI algorithm was trained to predict lung specific hot spot genomic alterations. An H & E stained slide was imaged with a 40x objective lens to obtain a brightfield image as an input data. The input image was segmented into 65 x 103 tiles, each of 64 µm2 in size for an individual pathological class. 80% of the data set was used for training the engine while validation and blinded study was respectively performed on remaining 10% data. Results: Using single blinded clinical samples. AI model analyzed whole slides. Table 1 summarize parameters for clinically significant variants. Higher sensitivity and specificity was observed for variants encoding mutated cell surface proteins compared to variants encoding intracellular mutant targets when compared on similar training and testing parameters. Conclusions: AI-enabled prediction of hotspot genomic alterations resulted comparable to NGS with shortest TAT, cost and resources. It predicted actionable gene alterations with high positive and negative prediction values. Table 1. Gene Sample (n = 480) Evaluation parameters for tested samples Training Testing Sensitivity (%) Specificity (%) PPV (%) NPV (%) F1-score (%) Accuracy (%) KRAS 248 62 100 94 94 100 97 97 ALK 16 4 100 100 100 100 100 100 EGFR 72 18 88 100 100 90 94 94 MET 16 4 50 100 100 66 66 75 ROS 16 4 100 100 100 100 100 100 STK11 88 22 82 73 75 80 78 77 RET 24 6 50 100 100 60 50 67 BRAF 14 2 100 100 100 100 100 100 NTRK 92 24 78 56 64 71 70 67 ERBB2 12 4 50 50 50 50 50 50 Citation Format: Gowhar Shafi, Shiva Shivamurthy, Anand Ulle, Chongtham Cha Chinglemba, Sumit Haldar, Fauzul Moubeen, Mohan Uttarwar. AI-enabled prediction of lung cancer specific hot spot gene alterations from histology images. [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 5368.

Abstract 1489: Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC)

Abstract The abnormality of ribosomal biogenesis is found to be involved in carcinogenesis and progression in various types of cancer. RPL22L1 gene is a highly homologous paralog of RPL22, both of which belong to the L22E family of ribosomal proteins and take part in encoding a cytoplasmic ribosomal protein that is a component of the 60S subunit of ribosomes. Our previous work has established a CRISPR/Cas9 genome-wide knockout screening profile and developed a two-component mixture model to identify cancer-specific essential genes in 436 cancer cell lines. Bioinformatics analysis reveals that RPL22L1 acts as a feature gene to predict cancer-specific essential genes in human cancer. Previous studies suggest that RPL22L1 is correlated with tumor progression and adverse prognosis in several cancer types, while its role in hepatocellular carcinoma (HCC) is unknown. In the present study, we found that RPL22L1 is highly expressed in embryonic stem cells (ES) and liver progenitor cells, which were acquired from a vitro hepatic differentiation model originating from ES cells. Hence, we hypothesized that RPL22L1 enhances cancer stem cell properties in HCC. Functional assays showed that RPL22L1 would promote sphere formation in vitro. qRT-PCR and Western Blotting results indicated that RPL22L1 could promote stemness-related gene expressions such as CD133, CD44, and SOX9 in HCC cell lines. Overexpression of RPL22L1 increased aldehyde dehydrogenase (ALDH) activity, which was decreased when RPL22L1 was silenced with short hairpin RNA (shRNA). Extreme limiting dilution assay showed that RPL22L1 overexpression stimulated self-renewal ability in immunodeficient mice. Furthermore, higher RPL22L1 expression can also promote cell proliferation and epithelial-mesenchymal-transition, as well as drug resistance to 5-Fluorouracil (5-FU) and cisplatin (CDDP). Silver nitrate staining demonstrated that RPL22L1 overexpression correlated with a larger size and number of nucleoli. The expression of RPL22L1 was also associated with adverse prognosis in HCC clinical samples. In conclusion, our study reveals aberrant ribosomal protein activity in HCC and the role of RPL22L1 in promoting HCC tumor progression. Citation Format: JinLin Huang, Zhenyang Guo, Jie Luo, Beilei Liu, Lanqi Gong, Xiaona Fang, Dora Lai-wan Kwong, Xinyuan Guan. Overexpression of ribosomal L22-like1 (RPL22L1) enhances cancer stem cell properties and promotes tumor progression in hepatocellular carcinoma (HCC) [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 1489.

Abstract 2076: Novel framework for systematically detecting alternative transcript initiation by integrating ATAC-seq and RNA-seq

Abstract Background: Alternative transcription initiation (ATI) has been frequently observed in cancer suggesting that it contributes to the malignant transformation of the cells. However, ATI remains largely unexplored mainly due to the lack of tools for detecting ATI. We propose a computational method for integrating bulk ATAC-seq and bulk RNA-seq to identify ATI and understand their differential usage between tissues. We hypothesize that differential ATAC-seq intensity can be used as a guide for looking for differential promoter usage, which might enable the identification of novel ATI events in a transcript-agnostic way. Methods: We recently published methods for RNA-seq base-level analysis for identifying structural variations in transcripts. Building on this, we developed a supervised sparse non-negative matrix factorization approach that integrates base-level RNA-seq and ATAC-seq, which aims to identify ATIs as well as characterize the latent structure of the underlying isoforms. This method equips many unique features that can be useful for the identification of novel ATIs. Because the method scans an entire collection of DNA accessible regions provided by ATAC-seq, it enables a comprehensive screening of novel ATI candidates that are not limited to the known promoters. Additionally, the uncompressed view of base-level RNA-seq allows us to infer the structure of individual isoforms independently of known gene annotation. The predicted isoforms can be further used to deconvolute base-level RNA-seq of individual cases into the isoforms and thus infer expression levels of each isoform. Results: We applied this method to a sub-cohort (N=350) of TCGA pan-cancer samples in which both ATAC-seq and RNA-seq are available. Empiric comparison to existing methods confirmed known true positive ATIs including important cancer genes such as CDKN2A and ALK. In particular, the method successfully identified the ATIs including some challenging cases such as ATIs located at internal introns or at constitutive exons in other transcripts. The deconvolution analysis applied to the extended cohort to all available ~10,000 TCGA pan-caner samples across 32 tissue types revealed that ATIs were predominantly differentiated across tissue types. By applying the method to a set of transcription regulator genes, we identified ~1% of the genes had ATIs including the novel cancer-specific genes with known ATIs that had not been previously reported as relevant to cancer. Additionally, we provide examples in which the isoform’s function relative to cancer appears mechanistically interesting. Conclusion: We propose a multi-omics integration method that is independent of known gene annotation, enabling a robust identification of ATIs. Our results strongly demonstrate our ability to pick up known as well as novel ATIs that are otherwise difficult to identify by existing methods. Citation Format: Hyo Young Choi, Won-Young Choi, David N. Hayes. Novel framework for systematically detecting alternative transcript initiation by integrating ATAC-seq and RNA-seq [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 2076.