Altered Gene Research Articles

Human DNA Mutations and their Impact on Genetic Disorders.

DNA is a remarkably precise medium for copying and storing biological information. It serves as a design for cellular machinery that permits cells, organs, and even whole organisms to work. The fidelity of DNA replication results from the action of hundreds of genes involved in proofreading and damage repair. All human cells can acquire genetic changes in their DNA all over life. Genetic mutations are changes to the DNA sequence that happen during cell division when the cells make copies of themselves. Mutations in the DNA can cause genetic illnesses such as cancer, or they could help humans better adapt to their environment over time. The endogenous reactive metabolites, therapeutic medicines, and an excess of environmental mutagens, such as UV rays all continuously damage DNA, compromising its integrity. One or more chromosomal alterations and point mutations at a single site (monogenic mutation) including deletions, duplications, and inversions illustrate such DNA mutations. Genetic conditions can occur when an altered gene is inherited from parents, which increases the risk of developing that particular condition, or some gene alterations can happen randomly. Moreover, symptoms of genetic conditions depend on which gene has a mutation. There are many different diseases and conditions caused by mutations. Some of the most common genetic conditions are Alzheimer's disease, some cancers, cystic fibrosis, Down syndrome, and sickle cell disease. Interestingly, scientists find that DNA mutations are more common than formerly thought. This review outlines the main DNA mutations that occur along the human genome and their influence on human health. The subject of patents pertaining to DNA mutations and genetic disorders has been brought up.

AI-Assisted Generation and In-depth In-silico Evaluation of Potential Inhibitor Targeting Aurora Kinase A (AURKA): An Anticancer Discovery Exploiting Synthetic Lethality Approach

Genetic alterations are lead causative agents behind the complex pathologies of cancers which render all treatments unarmed. Such alterations in oncogenes can be treated by direct inhibition by specific drugs while alteration in tumor suppressor genes mediating loss of function is challenging to treat. Identification of synthetic lethal partners to specific tumor suppressor genes and mediating their inhibition can be a potential approach to deal with loss of function mutations. Aurora kinase A (AURKA) has been established as an effective synthetic lethal partner of several tumor suppressor genes and is overexpressed in cancerous conditions, mediating adverse pathologies. The present AI-assisted study deals with the generation of novel inhibitor compounds against AURKA and the exhaustive evaluation of the best compound using molecular docking, molecular dynamic simulation, MM/PBSA, and QM/MMGBSA-based analysis. Out of the 200 novel compounds generated using features of ATP binding pocket of AURKA and previously reported inhibitor, compound 1 (4-{5-fluoro-6-[(1Z)-3-hydrazinyl-3-oxo-2-phenylprop-1-en-1-yl]pyridin-2-yl}benzoic acid) was identified as the most potent candidate with high negative binding energy of -10.4 kcal/mol in molecular docking analysis. The molecular dynamic simulation analysis of the AURKA-compound 1 complex and the apo-AURKA protein resulted in major conformational changes in the conserved DFG motif and loop 277-291 of AURKA in the AURKA-compound 1 complex thus inhibiting the entry of ATP to its binding site. The free energy landscape displayed a persistence of folded states of the enzyme in complex form. The MM/PBSA revealed effective Gibb’s free energy of binding of -11 kcal/mol for compound 1 inhibiting AURKA. The QM/MMGBSA analysis resulted in a significantly high negative binding energy of -13.98 kcal/mol proving significant inhibition potential of compound 1 against AURKA. Therefore, further in-vitro investigation can provide a novel effective, and safe treatment against a wide range of cancers by targeting a well-established cancer target AURKA.

Bioinformatic Analysis of Neuropeptide Related Genes in Patients Diagnosed with Invasive Breast Carcinoma

PurposeNeuropeptide receptors are expressed in many malignancies. Effectors involved in the action mechanisms of HCRTR1, HCRTR2, NPY4R (PPYR1) may be related to breast cancer (BC). Genes encoding these receptors and PPY and PTPN11 genes were aimed to examine via bioinformatics tools in the BRCA cohort. To our knowledge, this is the first study in which these receptor genes and PP, which have not found much research in BC, are examined together with PTPN11 and analyzed comprehensively in large patient cohorts from public databases. MethodscBioPortal was used for gene alteration analyses, GeneMania for association analyses with other genes, Kaplan-Meier Plotter for Overall Survival (OS) and Relapse Free Survival (RFS) analyses, UALCAN for methylation analyses, TIMER2.0 for expression analyses, The Human Protein Atlas database for expression validations, TIMER for immune infiltration analyses, WEKA 3.8.6 for diagnostic classification performances of the genes based on Random Forest Classifier and Enrichr-KG for Gene Ontology (GO) Biological Process (BP) and KEGG analysis. Results19 (1.9%) nucleotide changes were found in 996 cases. Missense mutation is most common. Decreased expression levels of the HCRTR1 gene were associated with shorter OS and RFS, but decreased expression levels of the PTPN11 gene were associated with longer OS and RFS. Decreased expression levels NPY4R (PPYR1) gene were associated with shorter RFS. Increased expression levels of HCRTR2 and PPY genes were associated with longer RFS. HCRTR1 and NPY4R (PPYR1) genes were statistically hypermethylated; conversely HCRTR2 and PPY genes were hypomethylated. There was no significant change in PTPN11 gene promoter methylation level. HCRTR1, NPY4R (PPYR1) and PTPN11 gene expressions were downregulated; conversely, HCRTR2 and PPY gene expressions upregulated. Weak correlations were observed between NPY4R (PPYR1) gene expression and CD4+ T Cell, Neutrophil, Dendritic Cell and between PTPN11 gene expression and CD8+ T Cell, Macrophage, Neutrophil, Dendritic Cell infiltrations. Area under the receiver operating characteristics curve values of the 10-fold cross-validation and by splitting the dataset in a ratio of 80:20 models were 0.930 and 0.963 respectively. HCRTR2 and HCRTR1 belong to regulation of cytosolic calcium ion concentration, cellular calcium ion homeostasis GO BPs. ConclusionIn BC patients, increases in HCRTR2 and PPY gene expressions could be considered as positive prognostic factors. Decreases in HCRTR1 and NPY4R (PPYR1) gene expressions could be considered as negative prognostic factors. Decreased expression of PTPN11 gene may have a positive prognostic factor. Changes in existing genes are likely to be both a biomarker and therapeutic target for BC. However, experimental and clinical studies are needed to elucidate the mechanisms underlying these neuropeptide receptors in terms of breast carcinogenesis.

Talazoparib plus enzalutamide in metastatic castration-resistant prostate cancer: Safety analyses from the randomized, placebo-controlled, phase III TALAPRO-2 study

This detailed analysis further characterizes the safety profile of talazoparib plus enzalutamide in the ongoing randomized, phase III TALAPRO-2 study in patients with metastatic castration-resistant prostate cancer (mCRPC). In both the all-comers and homologous recombination repair (HRR)-deficient populations, talazoparib plus enzalutamide significantly improved radiographic progression-free survival compared with placebo plus enzalutamide. The talazoparib plus enzalutamide safety populations in TALAPRO-2 included 398 patients from cohort 1 (all-comers, unselected for HRR gene alterations) and 198 patients from the combined HRR-deficient population (patients from the all-comers population with HRR gene alterations plus subsequently enrolled patients with HRR gene alterations; cohort 2). Patients received talazoparib 0.5mg (0.35mg, moderate renal impairment) and enzalutamide 160mg once daily. Safety analyses evaluated common treatment-emergent adverse events (TEAE), their type, severity, timing, seriousness, and relationship to study treatment. In the all-comers (n=398) and HRR-deficient populations (n=198), all-cause grade 3/4 (G3/4) TEAEs with talazoparib plus enzalutamide were reported in 71.9% and 66.2% of patients, respectively. Most common G3/4 hematologic TEAEs were anemia (46.7% and 40.9%, respectively), neutropenia (18.3% and 18.7%), and thrombocytopenia (7.3% and 7.1%). Median time to event was 3.3 and 3.3 months for G3/4 anemia, 2.3 and 2.3 months for G3/4 neutropenia, and 2.3 and 1.5 months for G3/4 thrombocytopenia. Maximum hemoglobin reduction occurred after 13 and 15 weeks of treatment. 18.8% and 10.1% of patients discontinued talazoparib. TEAEs were managed with dose interruption (62.1% and 57.6%), reduction (52.8% and 52.0%), hematologic supportive care (13.1% and 10.6%), and packed red blood cell transfusions (39.2% and 35.9%). Talazoparib plus enzalutamide had a generally manageable safety profile in patients with mCRPC within the all-comers and the HRR-deficient populations. NCT03395197.

Identifying genetic targets in clinical subtypes of Parkinson’s disease for optimizing pharmacological treatment strategies

Abstract The heterogeneity of Parkinson’s disease (PD) has been recognized in clinical, with patients categorized into distinct subsets based on motor phenotype, such as tremor-dominant PD (TD), postural instability and gait difficulty-dominant PD (PIGD) and mixed PD (Mix). Despite this categorization, the underlying mechanisms of this heterogeneity remain poorly understood, and there is no personalized effective treatment for each PD subtype. To address this, a rat model for PD subtypes was established by unilateral stereotaxic injection of 6-OHDA, followed by cluster analysis of behavioral data. The serum neurofilament light chain (NfL) and uric acid (UA) levels as well as alterations in brain autonomic activity in rats were consistent with clinical patients, and metabolomics results showed that more than 70% of the metabolites in the serum of different subtypes of PD rats and clinical patients appeared to be consistently altered. Further transcriptomic analysis by RNA-seq has elucidated that the development of PD subtypes is associated with altered gene expression in neurotransmitter, neuronal damage in the central or peripheral nervous system, and lipid metabolism. In addition, based on the subtype-specific differentially expressed genes, 25 potential drug candidates were identified. Notably, the Alox15 inhibitor baicalein showed a greater efficacy on Mix rats, highlighting the possibility of selecting targeted treatments for well-defined individuals.

Abstract A042: Desmosome mutations in the microenvironment regulate melanoma proliferation

Abstract Desmosomes are transmembrane protein complexes critical for cell-cell adhesion in epithelial tissues and other cell types. In skin cancers like melanoma, epithelial cells called keratinocytes are the predominant cells in the primary melanoma microenvironment, yet it remains largely unknown whether this cell type is subject to genetic alterations during melanoma development and its role in early melanoma progression. In this study, using an integrative analysis of tumor mutations and protein biophysical interactions, we identify a high frequency of genetic alterations in desmosome-related genes in human cancers, with the highest occurrences in cutaneous melanoma. Notably, over 70% of cutaneous melanoma cases exhibit mutations in desmosome genes and desmosome gene mutations are associated with lowered expression of the desmosome complex in melanoma. Using spatial transcriptomics and immunohistochemistry analyses, we find that the decrease in desmosome gene expression predominantly occurs in keratinocytes within the tumor microenvironment rather than in the melanoma cells themselves. To study the functional significance of this alteration in melanoma development, we used a human melanoma/keratinocyte co-culture system using primary melanoma cell lines. Our studies show that knockdown of desmosome genes in keratinocytes markedly increased proliferation of adjacent melanoma cells in melanoma/keratinocyte co-cultures. Additionally, melanoma cell proliferation is enhanced when exposed to media preconditioned by desmosome-deficient keratinocytes. These observations suggest that the gradual accumulation of mutations in desmosome genes within neighboring keratinocytes may create a conducive environment that primes melanoma cells for neoplastic transformation. This study underscores the critical role of genetic alterations in the tumor microenvironment in melanoma progression and highlights the potential for targeting desmosome-related pathways in therapeutic strategies against melanoma. Citation Format: Mohita Tagore. Desmosome mutations in the microenvironment regulate melanoma proliferation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A042.

High-grade B-cell lymphoma not otherwise specified, with diffuse large B-cell lymphoma gene expression signatures: Genomic analysis and potential therapeutics.

High-grade B-cell lymphoma not otherwise specified (HGBCL, NOS) has overlapping morphological and genetic features with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL), leading to uncertainty in its diagnosis and clinical management. Using functional genomic approaches, we previously characterized HGBCL and NOS, that demonstrate gene expression profiling (GEP), and genetic signatures similar to BL. Herein, we characterize distinct HGBCL, NOS, cohort (n = 55) in adults (n = 45) and in children (n = 10), and compared the GEP, genomic DNA copy number (CN), and mutational spectrum with de novo DLBCL (n = 85) and BL (n = 52). This subgroup, representing ~60% of HGBCL, NOS, lack gene-expression signature of BL and double hit/dark zone lymphoma, but express DLBCL like signatures and are characterized by either GCB- or ABC-like mRNA signatures and exhibit higher genomic complexity, similar to de novo DLBCL, and show alteration in genes regulating B-cell activation (CD79B, MYD88, PRDM1, TBLIXR1, CARD11), epigenome (KMT2D, TET2) and cell cycle transition (TP53, ASPM). However, recurrent mutations in genes often mutated in BL (DDX3X, GNA13, CCND3), but rare in DLBCL, are also present in HGBCL-NOS, highlighting genetic heterogeneity. Consistent with mutation spectrum, frequent genomic CN alterations in genes regulating B-cell activation (del-PRDM1, gain-BCL6, -REL, -STAT3) and cell cycle regulators (del-TP53, del-CDKN2A, del-RB1, gain-CCND3) were observed. Pediatric cases showed GCB-DLBCL-like mRNA signatures, but also featured hallmark mutations of pediatric BL. Frequent oncogenic PIM1 mutations were present in adult HGBCL, NOS. In vitro analyses with pharmacologic or genetic inhibition of PIM1 expression triggered B-cell activation and NF-κB-induced apoptosis, suggesting that PIM1 is a rational therapeutic target.

The microbiota-gut-brain axis: a potential target in the small-molecule compounds and gene therapeutic strategies for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by motor symptoms and non-motor symptoms. It has been found that intestinal issues usually precede motor symptoms. Microorganisms in the gastrointestinal tract can affect central nervous system through the microbiota-gut-brain axis. Accumulating evidence has shown that disturbances in the microbiota-gut-brain axis are linked with PD. Thus, this pathway appears to be a promising therapeutic target for treatment of PD. In this review, we mainly described gut dysbiosis in PD and their underlying mechanisms for mediating neuroinflammation and peripheral immune response in PD pathology and futher discussed the potential small-molecule compounds and genic therapeutic strategies targeting the microbiota-gut-brain axis and their applications in PD. Studies have found that some small molecule compounds and alterations of inflammation-related genes can improve the motor and non-motor symptoms of PD by improving the microbiota-gut-brain axis, which may provide potentially beneficial drugs and molecular targets for the therapies of PD.

CDH2 and CDH13 as potential prognostic and therapeutic targets for adrenocortical carcinoma

ABSTRACT Cadherin 2 (CDH2, N-cadherin) and cadherin 13 (CDH13, T-cadherin, H-cadherin) affect the progress and prognoses of many cancers. However, their roles in adrenocortical carcinoma (ACC), a rare endocrine cancer, remain unclear. To decipher the roles of these proteins in ACC and to identify their regulatory targets, we analyzed their expression levels, gene regulatory networks, prognostic value, and targets in ACC, using various bioinformatic analyses. CDH2 was strongly downregulated and CDH13 was strongly upregulated in patients with ACC; the expression levels of these genes affected the prognosis. In 75 patients, the expression of CDH2 and CDH13 was altered by 8% and 5%, respectively. CDH2 and CDH13, as well as their neighboring genes, were predicted to form a complex network of interactions, mainly through coexpression and physical and genetic interactions. CDH2 and its altered neighboring genes (ANGs) mainly affect tumor-related gene expression, cell cycle, and energy metabolism. The regulation of tumor-related integrin function, gene transcription, metabolism, and amide and phospholipid metabolism are the main functions of CDH13 and its ANGs. MiRNA and kinase targets of CDH2 and CDH13 in ACC were identified. CDH13 expression in patients with ACC was positively associated with immune cell infiltration. Anti-PD1/CTLA-4/PD-L1 immunotherapy significantly downregulated the expression of CDH13 in patients with ACC. Foretinib and elesclomol were predicted to exert strong inhibitory effects on SW13 cells by inhibiting the expression of CDH2 and CDH13. These data indicate that CDH2 and CDH13 are promising targets for precise treatment of ACC and may serve as new biomarkers for ACC prognosis.

Comprehensive genomic profiling for advanced hepatocellular carcinoma in clinical practice.

Although several therapeutic agents show efficacy in advanced hepatocellular carcinoma (HCC), biomarkers such as comprehensive genomic profiling (CGP) for the selection of second-line treatments after immunotherapy have not been established. We evaluated the value of CGP for the treatment decision in patients with HCC. We retrospectively studied 52 patients with advanced HCC who received CGP tests at three tertiary hospitals between February 2022 and November 2023. Genomic profiles were obtained using one of three CGP tests; 49 and 3 patients were evaluated using tissue-based and blood-based assay, respectively. The impact of CGP results on subsequent treatment selection in clinical practice and correlations between representative gene alterations and patient characteristics or responses to immunotherapy were evaluated. The most frequently observed variants were TERT mutations, followed by CTNNB1, TP53, ARID1A, and MYC mutations. Potentially druggable gene alterations were observed in 45 patients (87%), and 34 patients (65%) were recommended to receive treatments based on specific gene alterations by a molecular tumor board. Treatments were covered by health insurance in 13 patients (25%). Five patients (10%) received the recommended treatment by the date of data cut-off. There were no differences in the efficacy of immunotherapy with respect to mutation status in hTERT, CTNNB1, TP53, ARID1A, and MYC. The results of the present study suggested that druggable gene alterations may provide useful information not only in proposing alternative treatment after standard of care but also in selecting second-line targeted treatments after immunotherapy for patients with advanced HCC.

Abstract B022: Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs

Abstract Introduction: Extracellular vesicles have been found to be important regulators of intercellular communication between cancer cells. EV cargo varies greatly between different cell types, the composition of which provides important insights into the role of donor and recipient cells. Cetuximab Resistant Colorectal Cancer (CCCR) cells upregulate miR-100 and miR-125b and select them as cargo for their respective secreted EVs. These miRNAs are responsible for altered gene expression in the CCCR cells and can also be functionally transferred as part of EVs between donor and recipient cells. Bioinformatic analysis identified Cingulin (CGN) mRNA as one of the possible targets of miR-100 and miR-125b. The action of miRNAs leads to degradation of CGN mRNA ultimately decreasing cellular concentration of the functional protein. Absence of CGN has been associated with increased invasiveness and metastatic potential in the cancer cells. We carried out immunofluorescent imaging of CCCR cells which revealed downregulation of CGN in CCCR cells compared to the parental Cetuximab sensitive Colorectal Cancer (CC) cells. Similar effects were observed in recipient cells that obtained the miRNAs as part of EVs originating from CCCR donor cells. This project helps elucidate the roles of miR-100 and miR-125b in CCCR cells and how EVs facilitate their functional transfer between nearby cancer cells. Methods: CCCR cell lines were derived from parental CC cells in the lab of Dr. Robert Coffey by iterative selection of cetuximab resistance after growth in 3D. CRISPR/Cas9 technology was used to knockout miR-100 and miR-125b in CCCR cells. Transwell co-culture experiments were carried out to analyze EV transfer of miRNAs. Transfer was monitored by immunofluorescence using antibodies against CGN. Cells were stained and imaged as Z stacks under the 63x Immersion setting. ImageJ was used to analyze the stacks and CGN concentrations between the cells. For quantification of immunofluorescence, additional Z stack images were obtained from an inverted microscope and fluorescence digital camera. Images were analyzed using Fiji software to obtain specific CGN concentrations between the cells. Results and Discussion: Immunofluorescent imaging revealed statistically significant downregulation of CGN in CCCR cells. The protein was also downregulated in the recipient knockout cells as part of the Transwell co-culture experiment with CCCR donor cells. CGN is a tight junction protein and its absence confers increased invasiveness in 3D growth with enhanced metastasis. Decreased CGN concentration in the miR-100 and miR-125b enriched EV recipient knockout cells is indicative of functional transfer of the two miRNAs between cancer cells. These distinctive cellular dynamics could be harnessed to develop potential cancer therapies that provide a better prognosis and limit the metastatic potential of targeted cancer cells. Citation Format: Muhammad Shameer, Hannah M Nelson, Shimian Qu, Liyu Huang, Kevin C Corn, Sydney N Chapman, Nicole L Luthcke, Sara A Schuster, Tellie D Stamaris, Lauren A Turnbull, Lucas L Guy, Xiao Liu, Danielle L Michell, Elizabeth M Semler, Kasey C Vickers, Qi Liu, Jeffrey L Franklin, Alissa M Weaver, Marjan Rafat, Robert J Coffey, James G Patton. Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B022.

Integrative Transcriptomic and Metabolomic Analysis Reveals Quinoa Leaf Response Mechanisms to Different Phosphorus Concentrations During Filling Stage

Quinoa is an annual self-pollinating plant rich in polyphenols, flavonoids, saponins, and amino acids; its protein balance closely aligns with the ideal recommendation set by the Food and Agriculture Organization. Therefore, quinoa is considered the most suitable “all-nutrient food”. Phosphorus fertilization plays an important role in restricting the growth and development of quinoa; however, the effects of phosphorus fertilizer on quinoa growth remain unclear. Therefore, we conducted metabolome and transcriptome analyses on quinoa leaves during the filling stage, subjecting plants to different doses of phosphorus fertilizer. Overall, phosphorus treatment exerted a significant impact on the phenotypic characteristics of quinoa. Specifically, through a combined analysis of ultra-performance liquid chromatography–tandem mass spectrometry and transcriptome analysis, we identified the alteration and regulation of specific metabolites and genes within flavonoid biosynthesis pathways; this comprehensive evaluation helped elucidate the response mechanism of quinoa leaves during the grouting stage under various phosphorus conditions. Ultimately, the results of this study provide a reference for the selection of quinoa cultivars that exhibit tolerance to low- or high-phosphorus stress; additionally, we offer a theoretical basis for the rational application of phosphorus fertilizer and the enhancement of phosphorus utilization efficiency.

'Re-Wilding' an Animal Model With Microbiota Shifts Immunity and Stress Gene Expression During Infection.

The frequency of emerging disease is growing with ongoing human activity facilitating new host-pathogen interactions. Novel infection outcomes can also be shaped by the host microbiota. Caenorhabditis elegans nematodes experimentally colonised by a wild microbiota community and infected by the widespread animal pathogen, Staphylococcus aureus, have been shown to suffer higher mortality than those infected by the pathogen alone. Understanding the host responses to such microbiota-pathogen ecological interactions is key to pinpointing the mechanism underlying severe infection outcomes. We conducted transcriptomic analyses of C. elegans colonised by its native microbiota, S. aureus and both in combination. Correlations between altered collagen gene expression and heightened mortality in co-colonised hosts suggest the microbiota modified host resistance to infection. Furthermore, microbiota colonised hosts showed increased expression of immunity genes and variable expression of stress response genes during infection. Changes in host immunity and stress response could encompass both causes and effects of severe infection outcomes. 'Re-wilding' this model nematode host with its native microbiota indicated that typically commensal microbes can mediate molecular changes in the host that are costly when challenged by a novel emerging pathogen.

EXTH-30. GENETIC ALTERATIONS THAT DEREGULATE RB AND PDGFRA SIGNALING PATHWAYS DRIVE TUMOR PROGRESSION IN IDH2-MUTANT ASTROCYTOMA

Abstract In IDH-mutant astrocytoma, IDH2 mutation is quite rare and biological mechanisms underlying tumor progression in IDH2-mutant astrocytoma remain unclear. In this presentation, we report a unique case of IDH2 mutant astrocytoma, CNS WHO grade 3 that developed tumor progression. We performed a comprehensive genomic and epigenomic analysis for primary and recurrent tumors and found that both tumors harbored recurrent IDH2R172K and TP53R248W mutation with CDKN2A/B hemizygous deletion. We also found amplifications of CDK4 and MDM2 with PDGFRA gain in the recur- rent tumor and upregulated protein expressions of these genes. We further developed, for the first time, a xenograft mouse model of IDH2R172K and TP53R248W mutant astrocytoma from the recurrent tumor, but not from the primary tumor. Consistent with parent recurrent tumor cells, amplifications of CDK4 and MDM2 and PDGFRA gain were found, while CDKN2A/B was identified as homozygous deletion in the xenografts, qualifying for integrated diagnosis of astro- cytoma, IDH2-mutant, CNS WHO grade 4. Cell viability assay found that CDK4/6 inhibitor and PDGFR inhibitor potently decreased cell viability in recurrent tumor cells, as compared to primary tumor cells. On the other hand, in accord with our previous study that IDH1 inhibitor was not sufficient to induce anti-tumor effects in IDH1-mutant high-grade gliomas (Tateishi K et al. Cancer Cell. 2015), we did not find decreased cell viability nor histone methylation status changes after mutation specific IDH2 inhibitor treatment of recurrent cells. These findings underlie that gene alterations that activate retinoblastoma (RB) signaling pathways and PDGFR may drive tumor progression and xenograft formation in IDH2-mutant astrocytoma, which is equivalent to progressive IDH1-mutant astrocytoma. Also, our findings suggest that these genomic alterations may represent therapeutic targets in IDH2-mutant astrocytoma.

Compliance of a Tertiary Centre With Molecular Testing Strategies for Lynch Syndrome in Colorectal Cancer.

Introduction Lynch syndrome is one of the most common hereditary cancers associated with germline alterations of DNA mismatch repair genes. Recent advances have shed light on its molecular pathogenesis, leading to the development of various testing strategies. The aim of this study is to evaluate whether all colorectal adenocarcinomas undergo immunohistochemistry (IHC) and microsatellite instability (MSI) testing to identify potential Lynch syndrome patients. Methods This study evaluated the compliance of MSI and IHC testing for Lynch syndrome in a tertiary cancer research centre. All patients undergoing colorectal cancer resectionbetween April 2022 and December 2022were identified from a prospectively maintained register. The histology, MSI, and IHC testing reports for these patients were recorded to check if they were done and reported. Only patients with colorectal adenocarcinoma were included in this study. Results A total of 314 patients had colorectal cancer resections. A total of 301 were included in the study, with a median age of 65 years (IQR = 35-95). Thirteen (4.14%) patients did not have MSI and IHC testing reported on the system. Of these, eight (61.53%) did not have an MSI/IHC testing request, four (30.76%) did not have sufficient specimens to be sent for further testing, one (7.69%) had MSI and IHC testing but the final report was yet to be authorized. Out of the eight who did not have the testing done, three had early polyp cancer and one had a goblet cell adenocarcinoma. Conclusion According to the data, our hospital has a 97.45% compliance with current guidelines, not considering the insufficient sample for testing and the authorization of the final report. A more comprehensive proforma may be needed to provide a feedback loop for further MSI and IHC testing when entering data into the system. Further auditing is required to check the effectiveness of the proforma in achieving complete compliance.

TMIC-40. TARGETING GLIOMA TUMOR MICROENVIRONMENT BY SPECIFIC NEUTRALIZATION OF EXTRACELLULAR NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (ENAMPT) USING ALT-100, A NOVEL HUMANIZED MONOCLONAL ANTIBODY

Abstract BACKGROUND Intracellular nicotinamide phosphoribosyltransferase (iNAMPT is the rate-limiting enzyme of NAD salvage pathway and is preferentially used by glioma cells for regulating energy metabolism and homeostasis. When secreted, extracellular NAMPT (eNAMPT) exerts cytokine-like functions and is associated with poor prognosis in cancer patients. We have previously reported an essential role of iNAMPT for glioma cell proliferation and survival. Here, we examined whether eNAMPT influences glioma biology and tumor microenvironment by neutralizing eNAMPT using ALT-100, a novel humanized monoclonal antibody. METHODS Native levels of eNAMPT in blood samples of patients with primary/recurrent gliomas were determined using ELISA under an IRB approved protocol. Next, the effects of ALT-100 on glioma microenvironment were determined by treating viable human glioma tissue slices with ALT-100 and assessing downstream effects on RNA and protein using RNASeq, reverse phase protein array and spatial transcriptomics. Lastly, the in vivo effect of ALT-100-mediated eNAMPT neutralization on survival was determined in a mouse intracranial glioma model. RESULTS Measurable levels of eNAMPT were noted in all patient samples with a range of (3.7-71.2 ng/ml). Preliminary results showed a correlation of eNAMPT levels with grade of tumor. Human organotypic glioma slice cultures treated with ALT-100, showed a profound alteration of gene and protein expression particularly related to key energy metabolism and proliferation pathways and the PD-1/PD-L1 axis. Additionally, results of spatial profiling of ALT-100 treated human organotypic glioma slices will be presented. Survival and response data from ALT-100 treatment in ongoing in vivo experiment will also be reported. CONCLUSIONS This is the first report of ALT-100-mediated eNAMPT neutralization in gliomas and demonstrates profound effects on the tumor and its microenvironment. Given that ALT-100 is in phase 2 trials (NCT05938036) against ARDS, data from our studies can potentially accelerate translating ALT-100-mediated eNAMPT neutralization to treat gliomas.

Endogenous ZAP is associated with altered Zika virus infection phenotype

The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP.

Loss of CDKN2A Enhances the Efficacy of Immunotherapy in EGFR Mutant Non-Small Cell Lung Cancer.

Mutant epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). While mutant EGFR has been reported to limit the efficacy of immunotherapy, a subset of EGFR mutant NSCLC patients benefit from treatment with immune checkpoint inhibitors. A better understanding of how co-occurring genomic alterations in oncogenic driver genes impact immunotherapy efficacy may provide a more complete understanding of cancer heterogeneity and identify biomarkers of response. Here, we investigated the effects of frequent EGFR co-mutations in EGFR mutant lung cancer models and identified loss-of-function mutation of CDKN2A as a potential sensitizer to anti-PD-1 treatment in vitro and in vivo. Mechanistically, CDKN2A loss impacted the composition of the tumor immune microenvironment (TIME) by promoting the expression of PD-L2 through reduced ubiquitination of c-Myc, and mutant EGFR cooperated to upregulate c-Myc and PD-L2 by activating the MAPK pathway. Blocking PD-L2 induced anti-tumor immune responses mediated by CD8+ T cells in EGFR/CDKN2A co-mutated lung cancer. Importantly, a small-molecule PD-L2 inhibitor, zinc undecylenate, remodeled the TIME of EGFR/CDKN2A co-mutant tumors and enhanced the anti-tumor efficacy of EGFR-tyrosine kinase inhibitors. Collectively, these results identify EGFR/CDKN2A co-mutation as a distinct subtype of NSCLC that shows superior sensitivity to immune checkpoint blockade and reveals a potential combined therapeutic strategy for treating this NSCLC subtype.

Neutrophil Extracellular Traps Participate in the Pathogenesis of Lupus Through S100A10-Mediated Regulatory T-Cell Differentiation and Functional Abnormalities.

In systemic lupus erythematosus (SLE), neutrophil dysregulation and neutrophil extracellular traps (NETs) formation contribute to disease pathogenesis, potentially worsening the autoimmune response. Although research indicates NETs' involvement in various autoimmune conditions, their relationship with regulatory T cells (Tregs) in SLE remains elusive. In this study, in vivo experiments were involved in administering NET injections to C57BL/6 and MRL/Ipr mice. In vitro, a co-culture system facilitated interaction between Tregs and NETs. Proteomic analysis elucidated NET composition, while RNA sequencing delineated their impact on Treg differentiation. We demonstrated that increased NET levels correlate inversely with Treg abundance in SLE patients, influencing both their proportion and functionality. NET administration reduced Treg levels and induced lupus-like symptoms in C57BL/6 mice, exacerbating symptoms in MRL/Ipr mice. DNase I treatment mitigated NET effects, restoring Treg levels and alleviating symptoms. RNA sequencing revealed altered gene expression in naïve CD4+ T cells exposed to NETs. Additionally, proteomic analysis showed S100A10 protein changes between SLE patients and healthy controls, hindering Treg differentiation. NETs influence TLR-4 of naïve CD4+ T cells via S100A10, thereby modulating Treg proportion and functionality. These findings highlight the critical role of NETs in Treg differentiation in SLE, suggesting that targeting NETs may provide a novel therapeutic approach.

Histological transformation in lung adenocarcinoma: Insights of mechanisms and therapeutic windows.

Histological transformation from lung adenocarcinoma (ADC) to small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma (LCNEC), squamous cell carcinoma (SCC), and sarcomatoid carcinoma (PSC) after targeted therapies is recognized as a mechanism of resistance in ADC treatments. Patients with transformed lung cancer typically experience a poor prognosis and short survival time. However, effective treatment options for these patients are currently lacking. Therefore, understanding the mechanisms underlying histological transformation is crucial for the development of effective therapies. Hypotheses including intratumoral heterogeneity, cancer stem cells, and alteration of suppressor genes have been proposed to explain the mechanism of histological transformation. In this review, we provide a comprehensive overview of the known molecular features and signaling pathways of transformed tumors, and summarized potential therapies based on previous findings.