Cancer-associated Mutations Research Articles

Control of G protein–coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

Cryo-EM structure and biochemical analyses of the nucleosome containing the cancer-associated histone H3 mutation E97K.

The Lys mutation of the canonical histone H3.1 Glu97 residue (H3E97K) is found in cancer cells. Previous biochemical analyses revealed that the nucleosome containing the H3E97K mutation is extremely unstable as compared to the wild-type nucleosome. However, the mechanism by which the H3E97K mutation causes nucleosome instability has not been clarified yet. In the present study, the cryo-electron microscopy structure of the nucleosome containing the H3E97K mutation revealed that the entry/exit DNA regions of the H3E97K nucleosome are disordered, probably by detachment of the nucleosomal DNA from the H3 N-terminal regions. This may change the intra-molecular amino acid interactions with the replaced H3 Lys97 residue, inducing structural distortion around the mutated position in the nucleosome. Consistent with the nucleosomal DNA end flexibility and the nucleosome instability, the H3E97K mutation exhibited reduced binding of linker histone H1 to the nucleosome, defective activation of PRC2 (the essential methyltransferase for facultative heterochromatin formation) with a poly-nucleosome, and enhanced nucleosome transcription by RNA polymerase II.

Insulin and IGF-1 extend the lifespan of Caenorhabditis elegans by inhibiting insulin/insulin-like signaling and mTOR signaling pathways: C. elegans - Focused cancer research

The mutations in Caenorhabditis elegans (C. elegans) that extend lifespan slow down aging by interfering with several signaling pathways, including the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR). The tumor suppressor pRb (retinoblastoma protein) is believed to be involved in almost all human cancers. Lin-35, the C. elegans orthologue of the tumor suppressor pRb, was included in the study to explore the effects of insulin and IGF-1 because it has been linked to cancer-related pRb function in mammals and exhibits a tumor suppressor effect by inhibiting mTOR or IIS signaling. According to our results, IGF-1 or insulin increased the lifespan of lin-35 worms compared to N2 worms by increasing fertilization efficiency, also causing a significant increase in body size. It was concluded that the expression of daf-2 and rsks-1 decreased after insulin or IGF-1 administration, thus extending the lifespan of C. elegans lin-35 worms through both IIS and mTOR-dependent mechanisms. This suggests that it was mediated by the combined effect of the TOR and IIS pathways. These results, especially obtained in cancer-associated mutant lin-35 worms, will be useful in elucidating the C. elegans cancer model in the future.

Mutational profiling of 103 unresectable pancreatic ductal adenocarcinomas using EUS-guided fine-needle biopsy.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers, with a 5-year survival rate of around 9%. Only 20% are candidates for surgery. Most unresectable patients undergo EUS-guided fine-needle biopsy (EUS-FNB) for diagnosis. Identification of targetable mutations using next-generation sequencing (NGS) is increasingly requested. Data on feasibility of EUS-FNB for NGS and knowledge regarding mutational profile of unresectable PDAC are scarce. We evaluated the "technical yield" of EUS-FNB for NGS in unresectable PDAC: relative fraction of diagnostic EUS-FNBs meeting technical criteria. We also investigated the "molecular yield": relative fraction of EUS-FNBs included in NGS containing sufficient DNA for detection of at least one mutation. Furthermore, we determined the relative frequency of cancer-associated mutations in unresectable PDAC. Formalin-fixed and paraffin-embedded EUS-FNBs diagnostic of unresectable PDAC and fulfilling these criteria were included (n = 105): minimum 3-mm2 tissue, minimum of 2-mm2 tumor area, and minimum 20% relative tumor area. NGS was performed using Ion GeneStudio S5 Prime System and Oncomine™ Comprehensive Assay v.3 including 161 cancer-related genes. Technical yield was 48% (105/219) and molecular yield was 98% (103/105). Most frequently mutated genes were KRAS (89.3%) and TP53 (69.9%), followed by CDKN2A (24.3%), ARID1A (9.7%), SMAD4 (7.8%), TSC2 (7.8%), and CCND3 (6.8%). EUS-FNB for NGS of unresectable PDAC is feasible. Our technical criteria for NGS, using leftovers in formalin-fixed and paraffin-embedded blocks after routine pathology diagnosis, were met by around half of EUS-FNBs. Almost all EUS-FNBs fulfilling the technical criteria yielded a successful NGS analysis.

Integrative genomic analysis reveals cancer-associated mutations in patients with ophthalmic tumors.

Apart from the role of the retinoblastoma gene, the genomic events associated with poor outcomes in patients with ophthalmic tumors are poorly understood. We retrospectively analyzed 48 patients with six types of ophthalmic tumors. We searched for high-frequency mutated genes and susceptibility genes in these patients using combined exome and transcriptome analysis. We identified four clearly causative genes (TP53, PTCH1, SMO, BAP1). Susceptibility gene analysis identified hotspot genes, including RUNX1, APC, IDH2, and BRCA2, and high-frequency gene analysis identified several genes, including TP53, TTN, and MUC16. Transcriptome analysis identified 5868 differentially expressed genes, of which TOP2A and ZWINT were upregulated in all samples, while CFD, ELANE, HBA1, and HBB were downregulated. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the phosphoinositide 3-kinase (PI3K)-Akt and Transcriptional misregulation in cancer signaling pathways may be involved in ophthalmic tumorigenesis. TP53 is clearly involved in ophthalmic tumorigenesis, especially in basal cell carcinoma, and the PI3K-Akt signaling pathway may be an essential pathway involved in ophthalmic tumorigenesis. RUNX1, SMO, TOP2A, and ZWINT are also highly likely to be involved in ophthalmic tumorigenesis, but further functional experiments are needed to verify the mechanisms of these genes in regulating tumorigenesis.

Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing

Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

Cancer-associated mutations in protein kinase C theta are loss-of-function.

The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.

Abstract IA020: Functional studies of genetic variation using precision genome editing

Abstract Tumor genomes often harbor a complex spectrum of single nucleotide mutations, small indels, and large chromosome rearrangements that can perturb coding and non-coding regions of the genome in ways that remain poorly understood. The mutational processes responsible for the genesis of these events are also not static; instead, they continue to operate throughout disease evolution and further diversify the genetic and phenotypic landscape of cancer cells. Mounting evidence suggests that tumor genotype can be an important determinant of disease progression and therapy response, such as by modulating the sensitivity or resistance of cancer cells to mutant-specific drugs. These types of observations have motivated efforts to treat cancers with genome-informed therapies, highlighting the need to understand how different genetic variants precisely affect gene function and overall tumor phenotypes. Variant-function maps that provide a mechanistic understanding of the biology driven by cancer-associated mutations are needed to design these types of treatment strategies. Precision genome editing technologies like base and prime editing are uniquely suited to tackle this problem. Nevertheless, deploying these methods for systematic variant-function studies and disease modeling in vivo has not been straightforward due to lack of robust and scalable platforms capable of assessing editing efficiency and precision, particularly at endogenous loci. With this goal in mind, we previously developed and applied high-throughput base editing ‘sensor’ approaches that link endogenous genome editing outcomes with synthetic DNA-based readouts and cellular fitness measurements. Using these approaches, we showed that several uncharacterized mutant p53 alleles drive cancer cell proliferation and in vivo tumor development. Building upon this work, we recently developed new prime editing guide RNA design tools and sensor-based approaches that similarly couple quantitative editing outcomes to cellular fitness, allowing us to significantly expand the breadth of cancer-associated mutations that can be interrogated using these precision genome editing technologies. In this talk, I will describe ongoing work using base and prime editing sensor libraries to probe the biological impact of thousands of functionally-distinct genetic variants in diverse types of protein-coding genes and families to learn how these influence various cancer phenotypes. I will also discuss how the implementation of these modular technologies will allow researchers to functionally disentangle the impact of endogenous and exogenous mutational processes on functional selection and tumor evolution with close to single base pair resolution. This generalizable precision genome editing framework will facilitate the functional interrogation of genetic variants across diverse biological contexts, providing much needed insight into cancer variant-function relationships that could be leveraged to develop more precise cancer treatment paradigms, including synthetic lethal therapies that exploit tumor genotype. Citation Format: Francisco J. Sánchez-Rivera, Samuel I. Gould, Alexandra N. Wuest, Kexin Dong, Grace A. Johnson, Alvin Hsu, Varun K. Narendra, Ondine Atwa, Stuart S. Levine, David R. Liu. Functional studies of genetic variation using precision genome editing [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 IA020.

Barricades to aiding cascading: Patient-reported barriers to uptake in the context of clinician-facilitated cascade testing for hereditary cancer syndromes.

e13533 Background: Cascade testing extends genetic testing to relatives of individuals with known pathogenic variants. Cascade testing can prompt lifesaving cancer surveillance, however, up to two thirds of at-risk relatives do not complete recommended genetic testing. Previous studies suggest that a clinician-mediated approach of direct relative contact can increase testing rates. As part of an ongoing clinician-facilitated cascade testing intervention, we investigated the barriers to completion of cascade testing. Methods: All patients with a known cancer-associated germline mutation presenting to a gynecologic oncology clinic from 11/2023-2/2024 were offered clinician-facilitated cascade testing, an intervention whereby the genetics team was granted permission to contact designated at-risk relatives by telephone, review the familial mutation, and assist with accessing genetic testing. Patients that declined were asked about their decision and responses were analyzed using the socio-ecological model. Results: Ninety patients were offered clinician-facilitated cascade genetic testing and 50 (55%) declined. Of the patients that declined: median age was 45 (range 23-84), 41 (82%) self-identified as non-Hispanic, 38 (76%) as White, 23 (46%) were of Ashkenazi Jewish heritage, 23 (46%) had a personal history of cancer. There were no significant differences in any of the above variables when comparing those that declined and accepted the intervention. Twenty-nine (58%) patients declined because all family members were already tested. The most common barriers were “limited access to care” (5, 10%) at the healthcare community level, and “perceived readiness of relatives” (5, 10%) at the interpersonal relationship level. While these were the most common, most barriers were at the individual level including “perceived lack of responsibility” (3, 6%), “limited knowledge” (2, 4%), and “emotional burden” (2, 4%) (Table). Stated barriers were not associated with any demographical group. Conclusions: Although clinician-facilitated cascade testing has been shown to increase rates of relative testing uptake, several barriers persist at all levels of the socio-ecological model. While facilitated interventions should be individualized, our findings suggest that future interventions should focus efforts at all socio-ecological levels. Clinical trial information: NCT04613440 . [Table: see text]

Evaluation of concordance between blood-based and matched tissue molecular testing in a regional cancer center.

e15062 Background: Next-generation sequencing (NGS) to identify cancer-associated mutations together with an increasing array of targeted drugs with FDA approval allows for matching patients with more effective therapies. Tumor tissue molecular profiling is most often used to identify actionable biomarkers. Blood-based tests to sequence cell-free circulating tumor DNA (ctDNA) are a less invasive approach. Both methods have high specificity and sensitivity and provide insights into actionable biomarkers; however, little is known about how these NGS tests compare when used for the same patient. This study assessed the concordance of paired ctDNA and solid tumor profiling in patients treated in our regional cancer center. Methods: We retrospectively reviewed ctDNA and matching tumor sequencing data test results from CLIA/CAP laboratories between June 2018, and October 2023. The average testing turnaround time was 12.9 days for solid tumor sequencing and 7.7 days for ctDNA testing. We analyzed genomic variants, gene fusions, and microsatellite status in these patient results. The NGS tests for ctDNA included 83 relevant genes which reported single nucleotide variants, small insertions and deletions, splice variants, and limited copy number alterations and gene fusions. The NGS tests for tumor testing were performed on Formalin-Fixed Paraffin Embedded tissue samples using either a 592 relevant gene panel or whole exome sequencing, and either an RNA-based targeted capture panel of 52 genes or RNA whole transcriptome sequencing. Concordance is defined as both ctDNA and tumor testing reporting the same alterations, whereas discordant reports are defined as each reporting different alterations that didn’t overlap between ctDNA and tumor sequencing. Results: A total of 186 patients had both ctDNA and solid tumor sequencing tests done. Cancer types included pancreatic cancer (n = 42), breast cancer (n = 29), lung cancer (n = 28), colorectal cancer (n = 14), and others (n = 73). We observed concordance between tests in 76 (41%) patients in identifying the same genomic alterations. In 103 (55%) patients, we found discordant genomic findings (in the remaining 7 (4%) patients, there were no alterations detected (ND)). Among 103 patients for whom we found discordant findings between the tests, 41 (40%) showed genomic alterations only from the ctDNA test, 46 (45%) had results only from solid tumor profiling, and 16 (15%) had abnormal gene findings from both solid tumor and ctDNA testing but the results did not overlap. Conclusions: Our retrospective analysis comparing sequencing of ctDNA and tumor tissue demonstrated about 40% concordance in genomic findings. Our study indicates that these tests may be complementary. If one accepts a degree of discordance, then testing of ctDNA may be used to identify actionable genomic alterations in the setting of inaccessible or inadequate tumor samples for sequencing.

Structure and mechanism of the human CTDNEP1–NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.

Abstract A014: PIK3CA mutation might confer resistance to an FGFR inhibitor, erdafitinib, in urothelial cancer cells

Abstract Objective: Metastatic urothelial carcinoma (mUC) has a poor prognosis. In the United States, an FGFR inhibitor (erdafitinib) was approved for mUC patients with FGFR2/3 alterations. Significantly, FGFR3 mutations are associated with favorable outcomes in UC but might also be associated with less sensitivity to immune checkpoint inhibitors, especially in metastatic upper tract UC (mUTUC). Therefore, erdafitinib can be an optimal candidate for mUTUC with FGFR3 mutations before treatment with immune checkpoint inhibitors. However, the duration of response for erdafitinib is limited in many patients. Hence, this study aimed to elucidate the resistance mechanism of erdafitinib and develop a new treatment for mUC. Methods: UC cell lines with FGFR3 alterations were exposed to erdafitinib for an extended period to create resistant cell lines. We assessed genetic modifications in the resistant lines. We also investigated the susceptibility to specific therapeutic agents for cancer-associated gene mutations in vitro and in vivo. [Results] Whole exome sequencing demonstrated PIK3CA gene mutations in two resistant lines. Significantly, a PI3K inhibitor showed apoptosis in resistant cells in vitro. Moreover, alpelisib showed a statistically significant growth inhibitory effect on erdafitinib-resistant UC tumors in vivo compared to control or erdafitinib treatment. Conclusions: PIK3CA gene mutations may be one of the resistant mechanisms to erdafitinib and can also be targetable with a commercially available PI3K inhibitor in mUC. Citation Format: Seiji Arai, Tatsuhiro Sawada, Akira Ohtsu, Mai Onose, Yuta Maeno, Yoshitaka Sekine, Kazuhiro Suzuki. PIK3CA mutation might confer resistance to an FGFR inhibitor, erdafitinib, in urothelial cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A014.

Cascade genetic testing: an underutilized pathway to equitable cancer care?

The Precision Medicine Initiative was launched upon the potential of genomic information to tailor medical care. Cascade genetic testing represents a powerful application of precision medicine and involves the process of familial diffusion or the "cascade" of genomic risk information. When an individual (proband) is found to carry a cancer-associated germline pathogenic mutation, the information should be cascaded or shared with at-risk relatives. First degree relatives have a 50% likelihood of carrying the same cancer-associated mutation. This process of cascade testing offers at-risk relatives the opportunity for genetic testing and, for those who also carry the cancer-associated mutation, genetically targeted primary disease prevention through intensive cancer surveillance, chemoprevention and risk-reducing surgery, reducing morbidity and preventing mortality. Cascade testing has been designated by the Centers for Disease Control and Prevention as a Tier 1 genomic application for hereditary breast and ovarian cancer. In this manuscript we describe a cascade genetic testing and in particular focus on its potential to provide necessary care to medically underserved and vulnerable populations.

ASCC1 structures and bioinformatics reveal a novel helix-clasp-helix RNA-binding motif linked to a two-histidine phosphodiesterase

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE) (associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high resolution details and features conserved over 500 million years of evolution. Extending understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence–specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1 roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ.

Human DNA polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S using classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our findings suggest that Y432S induces structural alterations in domains responsible for nucleotide addition and ternary complex stabilization while retaining structural features consistent with possible catalysis in the active site. Calculations of the minimum energy path associated with the reaction mechanism of the wild type (WT) and Y432S Pol κ indicate that, while both enzymes are catalytically competent (in terms of energetics and the active site's geometries), the cancer mutation results in an endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on nonbonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3- over dCTP4-.

Phenotyping of cancer-associated somatic mutations in the BCL2 transmembrane domain

The BCL2 family of proteins controls cell death by modulating the permeabilization of the mitochondrial outer membrane through a fine-tuned equilibrium of interactions among anti- and pro-apoptotic members. The upregulation of anti-apoptotic BCL2 proteins represents an unfavorable prognostic factor in many tumor types due to their ability to shift the equilibrium toward cancer cell survival. Furthermore, cancer-associated somatic mutations in BCL2 genes interfere with the protein interaction network, thereby promoting cell survival. A range of studies have documented how these mutations affect the interactions between the cytosolic domains of BCL2 and evaluate the impact on cell death; however, as the BCL2 transmembrane interaction network remains poorly understood, somatic mutations affecting transmembrane regions have been classified as pathogenic-based solely on prediction algorithms. We comprehensively investigated cancer-associated somatic mutations affecting the transmembrane domain of BCL2 proteins and elucidated their effect on membrane insertion, hetero-interactions with the pro-apoptotic protein BAX, and modulation of cell death in cancer cells. Our findings reveal how specific mutations disrupt switchable interactions, alter the modulation of apoptosis, and contribute to cancer cell survival. These results provide experimental evidence to distinguish BCL2 transmembrane driver mutations from passenger mutations and provide new insight regarding selecting precision anti-tumor treatments.

Endometriosis, a common but enigmatic disease with many faces: current concept of pathophysiology, and diagnostic strategy.

Endometriosis is a benign, common, but controversial disease due to its enigmatic etiopathogenesis and biological behavior. Recent studies suggest multiple genetic, and environmental factors may affect its onset and development. Genomic analysis revealed the presence of cancer-associated gene mutations, which may reflect the neoplastic aspect of endometriosis. The management has changed dramatically with the development of fertility-preserving, minimally invasive therapies. Diagnostic strategies based on these recent basic and clinical findings are reviewed. With a focus on the presentation of clinical cases, we discuss the imaging manifestations of endometriomas, deep endometriosis, less common site and rare site endometriosis, various complications, endometriosis-associated tumor-like lesions, and malignant transformation, with pathophysiologic conditions.

Deconstructing cancer with precision genome editing.

Recent advances in genome editing technologies are allowing investigators to engineer and study cancer-associated mutations in their endogenous genetic contexts with high precision and efficiency. Of these, base editing and prime editing are quickly becoming gold-standards in the field due to their versatility and scalability. Here, we review the merits and limitations of these precision genome editing technologies, their application to modern cancer research, and speculate how these could be integrated to address future directions in the field.

Abstract P33: Novel Reporter Cell Models for Efficient Interrogation of Helicase Activity in Cancer-associated DDX3X Mutations

Abstract DDX3X is an ATP-dependent RNA helicase involved in diverse cellular processes. Somatic mutations in the DDX3X gene exhibit oncogenic or tumor suppressive activities depending on cancer types. However, helicase modulating roles of specific DDX3X mutations remain elusive mainly due to lack of an appropriate cell culture model system. Here, we report our development of novel transgenic cell lines capable of interrogating DDX3X helicase activity. To evaluate the helicase activity of the DDX3X mutations in question, we designed a reporter plasmid that measures the luciferase signal corresponding to the helicase activity. Using 8 different point mutations in the DDX3X that we identified earlier in relapsed/refractory patients with diffuse large B cell lymphoma (DLBCL), we show that these DDX3X mutants displayed defective helicase activity. DDX3X mutant knock-in DLBCL cells also displayed abolished helicase unwinding capabilities, validating our reporter cell models. We were able to extend the application of our reporter cells to interrogate Y chromosome-linked male paralog DDX3Y. In male lymphoma patients, ectopic DDX3Y expression can compensate for DDX3X loss to restore global protein synthesis. We narrowed down DDX3Y helicase and ATPase domains to be critical for cellular translation, ruling out DDX3Y involvement in ribosomal elongation. Unexpectedly, DDX3Y helicase is less efficient in RNA unwinding as compared to DDX3X revealing subtle differences despite having a high level of homology. The developed DDX3X reporter cells can be applied widely to evaluate the effects of DDX3X mutations in diseases as well as study the biology of DDX3X and DDX3Y. Citation Format: Zhi Sheng Poh, James Tan Chia Wei, Brandon Han Siang Wong, Kottaiswamy Amuthavalli, Suat Hoon Tan, Nicholas Francis Grigoropoulos, Navin Kumar Verma. Novel Reporter Cell Models for Efficient Interrogation of Helicase Activity in Cancer-associated DDX3X Mutations [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P33.