Mutational Spots Research Articles

Molecular Analysis of Renal/Adrenal Angiosarcomas Reveals High Frequency of Recurrent Genetic Alterations.

Angiosarcomas of the kidney and adrenal gland are rare, highly aggressive vascular neoplasms. Their genomic profile has not been systematically studied to date. We report the clinicopathologic and molecular features of six angiosarcomas centered in the kidney/adrenal gland. All patients were male adults, ranging from 58 to 77 years of age. Tumor sizes ranged from 2.5 to 22.5 cm. Half of the cases demonstrated hot spot mutations in the KDR gene, while one-third demonstrated mutations in the PIK3CA gene; both of these gene alterations being previously described, preferentially in breast angiosarcomas. In addition, two cases each demonstrated BRIP1 gene amplification, CTNNB1 and ETV6 mutations, which have not been previously reported in angiosarcoma. Notably, molecular studies were critical in establishing the correct diagnoses in three cases: one was an epithelioid angiosarcoma originally misdiagnosed as metastatic adenocarcinoma to the adrenal gland, the second was a vasoformative angiosarcoma that mimicked hemangioma, and the third was a collision tumor between a high-grade angiosarcoma and a chromophobe renal cell carcinoma which was originally diagnosed as a sarcomatoid renal cell carcinoma. In summary, angiosarcomas of the kidney and adrenal gland have a high frequency of recurrent genetic alterations, some of them being shared with other angiosarcoma subtypes, while other appear to be novel. In particular, activating hot spot KDR and PIK3CA mutations represent potential therapeutic targets for these highly aggressive cancers.

Novel method for detecting frequent TERT promoter hot spot mutations in bladder cancer samples

Telomerase reverse transcriptase promoter (TERTp) mutations are frequently targeted tumor markers, however, they reside in regions with high GC content, which poses challenges when examined with simple molecular techniques or even with next-generation sequencing (NGS). In bladder cancer (BC), TERTp mutations are particularly frequent, however, none of the available tools have demonstrated efficacy in detecting TERTp mutations via a simple noninvasive technique. Therefore, we developed a novel PCR-based method for the detection of the two most common TERTp mutations and demonstrated its use for the analysis of BC samples. The developed SHARD-PCR TERTp mutation detection technique requires PCR and restriction digestion steps that are easily implementable even in less well-equipped laboratories. Cell lines with known mutational status were utilized for method development. Matching urine and tumor tissue samples from BC patients were analyzed, and the results were validated by next-generation sequencing. Analysis of eighteen urine and corresponding tumor tissue samples by SHARD-PCR revealed perfect matches in sample pairs, which paralleled the corresponding NGS results: fourteen samples exhibited mutations at the −124 position, two samples showed mutations at the −146 position, and no mutations were detected in two samples. Our study serves as a proof-of-concept and is limited by its small sample size, nonetheless, it demonstrates that SHARD-PCR is a simple, economic and highly reliable method for detecting TERTp mutations, which are common in different cancer types. For bladder cancer, SHARD-PCR can be performed with the use of noninvasive samples and could replace or complement currently used techniques.

Evolutionary accumulation of FKS1 mutations from clinical echinocandin-resistant Candida auris

Introduction: Drug resistance to echinocandins, first-line drugs used to treat Candida auris infection, is rapidly emerging. However, the accumulation of mutations in genes other than FKS1 (before an isolate develops to resistance via FKS1 mutations), remains poorly understood. Methods Four clinical cases and 29 isolates associated with the incremental process of echinocandin resistance were collected and analyzed using antifungal drug susceptibility testing and genome sequencing to assess the evolution of echinocandin resistance. Findings: Six echinocandin minimum inhibitory concentration (MIC)-elevated C. auris strains and seven resistant strains were isolated from the urinary system of patients receiving echinocandin treatment. Meanwhile, phylogenetic analyses illustrated that the echinocandin-resistant strains were closely related to other strains in the same patient. Genomic data revealed that the echinocandin-resistant strains had FKS1 mutations. Furthermore, three categories (ECN-S/E/R) of non-synonymous mutant SNP genes (such as RBR3, IFF6, MKC1, MPH1, RAD2, and MYO1) in C. auris appeared to be associated with the three-stage-evolutionary model of echinocandin resistance in C. glabrata: cell wall stress, drug adaptation, and genetic escape (FKS mutation). Interpretation: Echinocandin-resistant C. auris undergoes spatial and temporal phase changes closely related to echinocandin exposure, particularly in the urinary system. These findings suggest that FKS1 mutations mediate an evolutionary accumulation of echinocandin resistance followed by modulation of chromosome remodeling and DNA repair processes that ultimately lead to FKS1 hot spot mutations and the development of drug resistance. This study provides an in-depth exploration of the molecular pathways involved in the evolution of Candida auris echinocandin resistance.

The Impact of Specific High-Pathogenic Tp53 Mutations on Survival in Head and Neck Cancers

Head and neck cancers (HNC) constitute about 30-40 % of all cancers in India. tp53 tumor suppressor gene is the most common gene mutated in HNC but has not been demonstrated to have any diagnostic or prognostic significance. therefore, we hypothesize that the survival rate and prognosis of head and neck cancer patients are linked with certain specific high-pathogenic tp53 hot spot mutations. To evaluate the hypothesis, we conducted a retrospective study on a cohort of 100 HNC patients between April 2013 and April 2018. targeted deep sequencing was performed to identify specific somatic mutations. Kaplan–Meier analysis and log-rank tests were performed. Tp53 mutations were identified in 47 of the 100 cases, with dual mutations in 4 patients. Functional characteristics displayed loss of function in 54.9% and gain of function in 27.5 % of cases. the majority of cases displayed missense aberration (74.5%), followed by nonsense mutations (11.76%). Of these pathogenic mutations, 33.3% fell into 6 hot spot residues while mini hot spot residues constituted 20.5% of the mutations. There was no statistically significant difference in the median survival rate between patients with and without the out tp53 mutation. however, the median survival rate was linked with poor prognosis in patients with specific pathogenic hot spot mutations in 247, 248, 245, and 175 residues (p value= 0.028). The majority of hot spot mutations (66%) showed a gain of function which may have been contributory to the pathogenicity of the disease. Results provide evidence that the existence of high-pathogenicity hot spot residues appears to be the only mutation that warrants further research to identify the therapeutic and prognostic significance of the tp53 gene in the treatment of HNC. further systemic studies are recommended to validate this hypothesis.

Development and Clinical Applications of Therapeutic Cancer Vaccines with Individualized and Shared Neoantigens.

Neoantigens, presented as peptides on the surfaces of cancer cells, have recently been proposed as optimal targets for immunotherapy in clinical practice. The promising outcomes of neoantigen-based cancer vaccines have inspired enthusiasm for their broader clinical applications. However, the individualized tumor-specific antigens (TSA) entail considerable costs and time due to the variable immunogenicity and response rates of these neoantigens-based vaccines, influenced by factors such as neoantigen response, vaccine types, and combination therapy. Given the crucial role of neoantigen efficacy, a number of bioinformatics algorithms and pipelines have been developed to improve the accuracy rate of prediction through considering a series of factors involving in HLA-peptide-TCR complex formation, including peptide presentation, HLA-peptide affinity, and TCR recognition. On the other hand, shared neoantigens, originating from driver mutations at hot mutation spots (e.g., KRASG12D), offer a promising and ideal target for the development of therapeutic cancer vaccines. A series of clinical practices have established the efficacy of these vaccines in patients with distinct HLA haplotypes. Moreover, increasing evidence demonstrated that a combination of tumor associated antigens (TAAs) and neoantigens can also improve the prognosis, thus expand the repertoire of shared neoantigens for cancer vaccines. In this review, we provide an overview of the complex process involved in identifying personalized neoantigens, their clinical applications, advances in vaccine technology, and explore the therapeutic potential of shared neoantigen strategies.

Abstract B009: Exploring the putative Kras-p53 mutational interface for vulnerability

Abstract Introduction: Kras Gain of Function mutations are frequently detected in lung, colorectal and pancreatic cancers, in addition to others. Activating Kras mutations results in a constantly active protein, which we reasoned is quite unphysiological, yet it is tolerated by cells since this mutation is seen in hyperplasia. Activating this oncogene is insufficient alone for malignant transformation. Mutations in the tumor suppressor gene (TSG) p53 (the most frequently mutated gene in human cancer), cooperate with mutant Kras and is sufficient to permit full display of the actions of oncogenic Ras (as confirmed in Genetically Engineered Mouse Models and in clinical cases). The phenomenon of oncogene addiction may in fact be the result of obligatory requirements (cellular metabolism or otherwise) brought on by the constant oncogene signaling. This required adjustment in cellular circuitry can be afforded by specific cooperating TSG and since it too is a DNA constant change, becomes an new rigid reality for a cell with a given set of oncogene-TSG pair. p53 mutational spectrum was described as ‘enigmatic’ presumably because they almost never completely abrogate the function of this major regulator suggesting to us a possible essential mechanistic role for the retained transcriptional targets within the (well annotated) p53 transcriptional network. This could represent a putative synthetic lethality opportunity against activated Kras, an oncogene that has proven difficult to drug. We hypothesized that specific mutations in p53 with their respective transcriptional lesions cooperate with unique mutant Kras in tissue specific manner identifying a short list of gene targets for synthetic lethality experiments. Methods: To examine this hypothesis, we analyzed the TCGA database to determine a conserved pair cooperation between common Kras 12C; D or V and the top 6 reported p53 (hot spot) mutations [on residues 175; 245;248;249;273 and 282] in a stage-agnostic manner. To examine whether those putative interactions are cell-type specific, we performed this analysis in 3 different histologies (lung, colon, and pancreas). Results: The results suggested a non-random distribution of p53 mutants among the Kras driven cancers. KRAS (12 C, 12 V, 12 D were reported in 80% of Colon, Pancreas, and Lung Cancer. p53 (175 - 30%) (245 - 30%) (248 - 15%) (249 - 12%) (273 - 6.5%) (282 - 6.5%). More detailed analysis is planned for the poster session. Conclusion: Despite having a single activated oncogene, the distribution of the cooperating p53 mutations is nonrandom so examining the transcriptionally retained gene list represents a novel approach to explore for gene editing experiments. This provides and approach to drug the addiction of cancers to their oncogenes in a cancer specific, and occasionally to target essential proto-oncogene such as Myc where direct inhibition is highly undesirable due to its physiologic roles. Citation Format: Nishanth Thalambedu, Shallya Anand, Haya Safar, Farah Mazahreh, Ahmad Mazin M. Safar. Exploring the putative Kras-p53 mutational interface for vulnerability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B009.

Mutation spectrum of hearing loss patients in Northwest China: Identification of 20 novel variants.

Hearing loss (HL) is the most frequent sensory deficit in humans, with strong genetic heterogeneity. The genetic diagnosis of HL is very important to aid treatment decisions and to provide prognostic information and genetic counselling for the patient's family. We detected and analysed 362 Chinese non-syndromic HL patients by screening of variants in 15 hot spot mutations. Subsequently, 40 patients underwent further whole-exome sequencing (WES) to determine genetic aetiology. The candidate variants were verified using Sanger sequencing. Twenty-three carrier couples with pathogenic variants or likely pathogenic variants chose to proceed with prenatal diagnosis using Sanger sequencing. Among the 362 HL patients, 102 were assigned a molecular diagnosis with 52 different variants in 22 deafness genes. A total of 41 (11.33%) cases with the biallelic GJB2 (OMIM # 220290) gene mutations were detected, and 21 (5.80%) had biallelic SLC26A4 (OMIM # 605646) mutations. Mitochondrial gene (OMIM # 561000) mutations were detected in seven (1.93%) patients. Twenty of the variants in 15 deafness genes were novel. SOX10 (OMIM # 602229), MYO15A (OMIM # 602666) and WFS1 (OMIM # 606201) were each detected in two patients. Meanwhile, OSBPL2 (OMIM # 606731), RRM2B (OMIM # 604712), OTOG (OMIM # 604487), STRC (OMIM # 606440), PCDH15 (OMIM # 605514), LOXHD1 (OMIM # 613072), CDH23 (OMIM # 605516), TMC1 (OMIM # 606706), CHD7 (OMIM # 608892), DIAPH3 (OMIM # 614567), TBC1D24 (OMIM # 613577), TIMM8A (OMIM # 300356), PTPRQ (OMIM # 603317), SALL1 (OMIM # 602218), and GSDME (OMIM # 608798) were each detected in one patient. In addition, as regards one couple with a heterozygous variant of CDH23 and PCDH15, respectively, prenatal diagnosis results suggest that the foetus had double heterozygous (DH) variants of CDH23 and PCDH15, which has a high risk to cause ID/F type Usher syndrome. Our study expanded the spectrum of deafness gene variation, which will contribute to the genetic diagnosis, prenatal diagnosis and the procreation guidance of deaf couple. In addition, the deafness caused by two genes should be paid attention to in the prenatal diagnosis of families with both deaf patients.

Prognostic genomic signatures in patients with RAS and BRAF wild-type (wt) metastatic colorectal cancer (mCRC) from the phase III study of first-line FOLFIRI/cetuximab versus FOLFIRI/cetuximab followed by cetuximab (Cet) alone (ERMES study).

3590 Background: The ERMES trial did not show non-inferiority of maintenance with Cet alone versus standard treatment. However, preliminary results suggested that a strategy of de-escalation treatment with only Cet might be effective in selected patients. Here we describe preliminary data from comprehensive genomic profiling (CGP) of CRC samples from the ERMES study. Methods: Patients with untreated RAS/BRAF wt mCRC were randomly assigned (1:1) to receive either FOLFIRI/Cet until PD/toxicity (arm A) or FOLFIRI/Cet for 8 cycles followed by Cet alone (arm B). DNA from tumor tissue samples was analyzed with the Oncomine Comprehensive Assay Plus covering hot spot mutations in over 500 genes. Variant calling was performed using the Ion Reporter v5.20 software. The prognostic value of genomic signatures was assessed in the intention-to-treat (ITT) patients’ population with available sequencing data. Results: CGP has been successfully completed so far for 139 patients of the ITT population (68 in arm A and 71 in arm B). This subgroup had a median progression free survival (mPFS) of 9.3 months. The comparison of the genomic profile of cases within the 25th and the 75th mPFS percentile (34 and 36 patients, respectively), identified two signatures that were defined: RES, 14 genes with genomic alterations unique to the 25th percentile poor prognosis group; SENS, 34 genes altered only in the 75th percentile good prognosis cohort. The RES signature was significantly associated with ERBB2, IL-4 and IL-13, MET activated PI3K/AKT, ESR, PI3K and FGFR4 signalling. When the RES signature was applied to the whole cohort, patients with at least one genomic alterations in any gene of the signature showed a mPFS of 2.4 months versus 15.2 months of the wt (Hazard Ratio, HR wt versus mutant 0.07). The SENS signature was associated with pathways related to gene transcription and expression, SMAD 2/3/4 transcription, DNA repair, chromatin modification and Activin signalling. The mPFS of SENS mutant and wt patients was 16.2 and 3.2 months, respectively (HR wt versus mutant 10.09). An exploratory analysis found no significant differences in mPFS for patients with good prognosis based on genomic signatures enrolled in the standard arm (A) versus the experimental arm (B). Conclusions: These preliminary data suggest that CGP of mCRC patients can identify prognostic genomic signatures, which might allow a better stratification of patients and the identification of subgroups that might benefit a treatment de-escalation strategy.

Clinical relevance of SCN and CyN induced by ELANE mutations: a systematic review.

According to the PRISMA criteria, a systematic review has been conducted to investigate the clinical relevance between patients with severe congenital neutropenia (SCN) and cyclic congenital neutropenia (CyN) induced by ELANE mutations. We have searched PubMed, EMBASE, Web of Science, Scopus, Cochrane, CNKI, Wanfang Medicine, and VIP for ELANE mutation related literature published from 1997 to 2022. Using Microsoft Excel collect and organize data, SPSS 25, GraphPad Prism 8.0.1, and Omap analyze and plot statistical. Compare the gender, age, geography, mutation sites, infection characteristics, treatment, and other factors of SCN and CyN patients induced by ELANE mutations, with a focus on exploring the relationship between genotype and clinical characteristics, genotype and prognosis. This study has included a total of 467 patients with SCN and 90 patients with CyN. The onset age of SCN and CyN are both less than 1 year old, and the onset and diagnosis age of SCN are both younger than CyN. The mutation of ELANE gene is mainly missense mutation, and hot spot mutations include S126L, P139L, G214R, c.597+1G>A. The high-frequency mutations with severe outcomes are A57V, L121H, L121P, c.597+1G>A, c.597+1G>T, S126L, C151Y, C151S, G214R, C223X. Respiratory tract, skin and mucosa are the most common infection sites, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli are the most common. Patients with refractory G-CSF are more likely to develop severe outcomes. The commonly used pre-treatment schemes for transplantation are Bu-Cy-ATG and Flu-Bu-ATG. The prognosis of transplantation is mostly good, but the risk of GVHD is high. https://www.crd.york.ac.uk/PROSPERO/. PROSPERO, identifier CRD42023434656.

Effect of DNMT3A R882H Hot Spot Mutations on DDX43 Promoter Methylation in Acute Myeloid Leukemia.

Epigenetic alterations have been observed in many hematological malignancies, including acute myeloid leukemia (AML). Many of these alterations result from mutations in DNA methyl transferase (DNMT) enzymes, disabling them to methylate target genes in a proper way. In this case-control study, we investigated the association between R882H mutation in DNMT3A gene and DDX43 gene methylation in patients with AML. 47 AML patients and 6 controls were included in this study. After DNA extraction, amplification refractory mutation system (ARMS)-PCR was used to evaluate R882H mutations in DNMT3A gene. The high-resolution melting (HRM) method was used to determine the methylation changes of the DDX43 gene promoter. R882H mutation was only found in 10.6% (5 out of 47) of AML patients. The frequency of DDX43 gene methylation was significantly higher in patients without R882H mutations compared to patients with R882H mutations (P < 0.05). The DNMT3A R882H mutation is typically present in a minority of AML patients. Nevertheless, this mutation is associated with a reduced frequency of methylation in the DDX43 promoter region.

The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA has acquired resistance to next-generation β-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators, which, through poorly described mechanisms, enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased β-lactam MICs up to 16-fold and transformed MRSA cells with low levels of resistance to being homogenously highly resistant to β-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPR interference (CRISPRi) to knock down spx transcription, we unambiguously linked hyper-resistance to the accumulation of Spx. Spx was previously proposed to be essential; however, our data suggest that Spx is dispensable for growth at 37°C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in β-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane, including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox-sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum β-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

Molecular mechanisms behind the generation of pro-oncogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s), characterized by amino acid insertions at the COOH-terminal region of the viral protein, have been recently identified and studied for their biological activity. Different from their wild-type counterpart (refp17), vp17s display a potent B cell growth and clonogenic activity. Recent data have highlighted the higher prevalence of vp17s in people living with HIV-1 (PLWH) with lymphoma compared with those without lymphoma, suggesting that vp17s may play a key role in lymphomagenesis. Molecular mechanisms involved in vp17 development are still unknown. Here we assessed the efficiency of HIV-1 Reverse Transcriptase (RT) in processing this genomic region and highlighted the existence of hot spots of mutation in Gag, at the end of the matrix protein and close to the matrix-capsid junction. This is possibly due to the presence of inverted repeats and palindromic sequences together with a high content of Adenine in the 322-342 nucleotide portion, which constrain HIV-1 RT to pause on the template. To define the recombinogenic properties of hot spots of mutation in the matrix gene, we developed plasmid vectors expressing Gag and a minimally modified Gag variant, and measured homologous recombination following cell co-nucleofection by next-generation sequencing. Data obtained allowed us to show that a wide range of recombination events occur in concomitance with the identified hot spots of mutation and that imperfect events may account for vp17s generation.

Complete Genome Sequencing, Annotation, and Mutational Profiling of the Novel Clade I Human Mpox Virus, Kamituga Strain.

Human Mpox (formerly monkeypox) infection is an emerging zoonotic disease caused by the Mpox virus (MPXV). We describe the complete genome annotation, phylogeny, and mutational profile of a novel, sustained Clade I Mpox outbreak in the city of Kamituga in Eastern Democratic Republic of the Congo (DRC). A cross-sectional, observational, cohort study was performed among patients of all ages admitted to the Kamituga Hospital with Mpox infection symptoms between late September 2023 and late January 2024. DNA was isolated from Mpox swabbed lesions and sequenced followed by phylogenetic analysis, genome annotation, and mutational profiling. We describe an ongoing Clade I Mpox outbreak in the city of Kamituga, South Kivu Province, Democratic Republic of Congo. Whole-genome sequencing of the viral RNA samples revealed, on average, 201.5 snps, 28 insertions, 81 deletions, 2 indels, 312.5 total variants, 158.3 amino acid changes, 81.66 intergenic variants, 72.16 synonymous mutations, 106 missense variants, 41.16 frameshift variants, and 3.33 inframe deletions across six samples. By assigning mutations at the proteome level for Kamituga MPXV sequences, we observed that seven proteins, namely, C9L (OPG047), I4L (OPG080), L6R (OPG105), A17L (OPG143), A25R (OPG151), A28L (OPG153), and B21R (OPG210) have emerged as hot spot mutations based on the consensuses inframe deletions, frameshift variants, synonymous variants, and amino acids substitutions. Based on the outcome of the annotation, we found a deletion of the D14L (OPG032) gene in all six samples. Following phylogenetic analysis and whole genome assembly, we determined that this cluster of Mpox infections is genetically distinct from previously reported Clade I outbreaks, and thus propose that the Kamituga Mpox outbreak represents a novel subgroup (subgroup VI) of Clade I MPXV. Here we report the complete viral genome for the ongoing Clade I Mpox Kamituga outbreak for the first time. This outbreak presents a distinct mutational profile from previously sequenced Clade I MPXV oubtreaks, suggesting that this cluster of infections is a novel subgroup (we term this subgroup VI). These findings underscore the need for ongoing vigilance and continued sequencing of novel Mpox threats in endemic regions.

Genotypic and phenotypic features of 39 Chinese patients with glycogen storage diseases type I, VI, and IX.

Glycogen storage diseases (GSDs) are abnormally inherited glycogen metabolism mainly affecting the liver, muscles, and heart. Deficiency of proteins involved in glycogen metabolism caused by genetic mutations are responsible for different subtype of GSDs. However, there are still some challenges in diagnosing GSD. This study includes 39 suspected GSDs patients from unrelated families in China. Next-generation sequencing (NGS) was used to investigate the reason for their diseases at the genetic level. Finally, all 39 patients were diagnosed with GSDs, including 20 GSD-Ia, 4 GSD-VI, and 15 GSD IX (12 GSD-IXa patients and 3 GSD-IXb patients). Thirty-two mutations in G6PC1, PYGL, PHKA2, and PHKB genes were identified, with 14 of them being novel variants. The pathogenicity of novel variants was classified according to ACMG guildlines and predicted by in slico algorithms. Mutations p.L216L and p.R83H in G6PC1 gene may be the hot spot mutation in Chinese. Hearing impairment is a rare clinical feature of GSD Ia, which has also been observed in our cohort. The severity of GSD VI and IX was indicated by our patients. Close follow-up should be applied to GSD VI and IX patients. Our findings provided evidence for building the phenotype-genotype of GSDs and expanded the mutation spectrum of related genes.

Abstract 4099: AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity

Abstract Background p53 is the most commonly mutated gene in human cancer, prevalent in nearly all tumor types. Despite its significant medical relevance and the knowledge that human T cells recognize these mutations[1,2], immunotherapies designed to promote responses against mutated p53 (mp53) have not had the hoped-for clinical impact[3]. The Amphiphile (AMP) platform improves the potency of vaccine immunotherapy by programming the delivery of vaccine components to the lymph nodes where efficient uptake by immune cells initiates tumor-targeted immune responses. AMP-modification of vaccine components (peptide antigens, molecular adjuvants) results in covalent conjugation to albumin-binding lipids. Upon injection, AMP-vaccines associate with tissue-resident albumin which efficiently distributes to draining lymph nodes. This approach was shown to promote activation of polyfunctional, cytotoxic T cells with promising safety and improved clinical outcomes in a Phase 1 trial of ELI-002, an mKRAS AMP therapeutic vaccine (AMPLIFY-201 NCT05726864). Application of this strategy to p53 mutations with ELI-008 offers the potential for improved immunotherapeutic activity in a setting of significant unmet need. Methods Following immunization of C57BL/6J mice with AMP-modified or soluble comparator vaccines consisting of mp53 peptides and CpG adjuvant, analyses were performed 7 days after the third bi-weekly dose. To assess antigen-specific T cell responses, ELISpot (IFNγ, GzmB), multiplexed proteomic, and flowcytometric analysis of effector cytokines (IFNγ, TNFα, IL-2) were performed following antigenic stimulation. Responses were determined in secondary lymphoid tissues and lung. Cytolytic capabilities of antigen-specific T cells were evaluated by monitoring specific killing of IV-transferred antigen-pulsed target cells. Results AMP-immunization generated robust immune responses yielding strong T cell activation against common p53 hot spot mutations (R248W, R248Q, R175H, G245S, R273H, Y220C, C135Y, R158H, H214R). Responses to AMP-vaccination were characterized by the generation of increased frequency of polyfunctional T cells (IFNγ, TNFα, IL2) specific to mp53 epitopes. Induced T cells demonstrated significant levels of cytolytic activity including GzmB production and elimination of target cells in vivo. Non-lymph targeted vaccines using soluble comparators were inactive. Conclusions AMP-conjugation permits efficient delivery directly to the lymph nodes and thus improves the immunogenicity of peptide vaccination. These substantially improved immune responses induced by ELI-008 represent a promising therapeutic opportunity for targeting cancer in a large fraction of human tumors. The AMP-platform technology is simple, rapid and scalable, promising broad clinical, off-the-shelf application for treating p53 mutated tumors. Citation Format: Martin P. Steinbuck, Xavier Cabana-Puig, Erica Palmer, Mimi M. Jung, Thomas Williams, Kristen Osaer, Jeff Zhang, Christopher M. Haqq, Peter C. DeMuth. AMP-peptide vaccination against multiple p53 mutant epitopes promotes lymph node delivery to generate potent, functional T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4099.

Abstract 1215: DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations

Abstract RAS mutations are found in 30% of human cancers and are characterized by hotspot mutations at codons G12, G13, and Q61. This results in constitutively activated RAS proteins. Despite recent advances in drug design and the emergence of covalent inhibitors, effectively targeting mutant RAS remains challenging. Our group previously identified the tumor suppressor protein DIRAS3, which directly binds Ras and inhibits its function by disrupting KRAS dimers/nanoclusters and blocking effector activation. DIRAS3 expression reduced cancer cell growth in pancreatic and ovarian cancers. To determine whether DIRAS3 plays a definitive role in inhibiting mutant KRAS, we comprehensively characterized DIRAS3's RAS-inhibitory effects using RASless mouse embryo fibroblast (MEF) cells as a model system lacking endogenous RAS isoforms. We ectopically expressed various KRAS mutations in these cells and then examined DIRAS3's effect on each KRAS mutant clone. We analyzed downstream RAS signaling, colony formation, cell viability, and toxicity. Our results showed that DIRAS3 significantly inhibited RAS-dependent colony formation in KRAS mutant (G12C, G12D, G12V, G13D, G12R, Q61R, Q61L) but not wild-type KRAS clones. However, when we added epidermal growth factor (EGF) to the medium, DIRAS3 expression inhibited wild-type KRAS activity, decreasing colony formation. This suggests that DIRAS3 selectively affects the active, GTP-bound form of KRAS and downstream signaling. Additionally, DIRAS3 expression decreased Erk1/2 phosphorylation in MEF cells with mutant KRAS, but not in the cells with a BRAFV600E mutation. This indicates that DIRAS3 specifically inhibits the KRAS-mediated MEK/ERK signaling, cell proliferation, and cytotoxicity. Moreover, to determine the critical region for DIRAS3's RAS-inhibitory function, we compared wild-type DIRAS3 and deletions (ΔNTE, ΔCTE, ΔNCTE). Our results showed both termini are required to effectively suppress clonogenic growth, emphasizing the requirement for DIRAS3 to anchor to the plasma membrane and interact with the RAS dimer interface to inhibit RAS-dependent tumor growth. In conclusion, our findings underscore the potent and specific inhibitory effects of DIRAS3 on RAS-driven oncogenesis, positioning it as a promising pan-RAS inhibitor for RAS-mutant cancers. Citation Format: Gamze Bildik, Junchen Liu, Weiqun Mao, Hailing Yang, John F. Hancock, Robert C. Bast, Zhen Lu. DIRAS3 inhibits oncogenic RAS signaling and RAS-dependent cell growth driven by prevalent KRAS hot spot mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1215.

Mitochondrial tRNASer(UCN) mutations associated non-syndromic sensorineural hearing loss in Chinese families

Mitochondrial transfer RNA mutation is one of the most important causes of hereditary hearing loss in humans. Mitochondrial transfer RNASer (UCN) gene is another hot spot for mutations associated with non-syndromic hearing loss, besides the 12S ribosomal RNA gene. In this study, we assessed the clinical phenotype and the molecular characteristics of two Chinese families with non-syndromic hearing loss. Mutational analysis revealed that 7445A > G and 7510T > C mutations in the mitochondrial transfer RNASer (UCN) gene were the molecular etiology of Family 1 and Family 2, respectively. However, the clinical and genetic characteristics of the two families carrying the above mutations in the transfer RNASer (UCN) gene exhibited a variable expression of hearing loss and an incomplete penetrance. Sequencing analysis of the complete mitochondrial genome showed the presence of transfer RNATrp 5568A > G and NADH-ubiquinone oxidoreductase chain 4 11696G > A mutations in Family 1. The mitochondrial haplotype analysis showed that the two families belonged to Asian D4 and M80′D haplotypes, respectively, and no pathogenic variations were found in the nuclear genes. To our knowledge, our study is the first to report 7445A > G and 7510T > C mutations in the mitochondrial transfer RNASer (UCN) gene, in multi-generation non-syndromic hearing loss pedigrees from China. Our study suggests that 5568A > G and 11696G > A mutations may enhance the penetrance of hearing loss in Chinese Family 1, while mitochondrial haplotypes and known nuclear genes may not be modifiers for the phenotypic expression of 7445A > G and 7510T > C mutations in these Chinese families.

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Predicting hotspots for disease-causing single nucleotide variants using sequences-based coevolution, network analysis, and machine learning.

To enable personalized medicine, it is important yet highly challenging to accurately predict disease-causing mutations in target proteins at high throughput. Previous computational methods have been developed using evolutionary information in combination with various biochemical and structural features of protein residues to discriminate neutral vs. deleterious mutations. However, the power of these methods is often limited because they either assume known protein structures or treat residues independently without fully considering their interactions. To address the above limitations, we build upon recent progress in machine learning, network analysis, and protein language models, and develop a sequences-based variant site prediction workflow based on the protein residue contact networks: 1. We employ and integrate various methods of building protein residue networks using state-of-the-art coevolution analysis tools (RaptorX, DeepMetaPSICOV, and SPOT-Contact) powered by deep learning. 2. We use machine learning algorithms (Random Forest, Gradient Boosting, and Extreme Gradient Boosting) to optimally combine 20 network centrality scores to jointly predict key residues as hot spots for disease mutations. 3. Using a dataset of 107 proteins rich in disease mutations, we rigorously evaluate the network scores individually and collectively (via machine learning). This work supports a promising strategy of combining an ensemble of network scores based on different coevolution analysis methods (and optionally predictive scores from other methods) via machine learning to predict hotspot sites of disease mutations, which will inform downstream applications of disease diagnosis and targeted drug design.

Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea

Purpose Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs. Methods The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits. Results We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age. Conclusion This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.