Chromosomal Instability Research Articles

Abstract 5047: Identifying genomic features associated with pathologic lymph node metastasis in lung adenocarcinoma patients

Abstract Introduction: Lymph node metastasis evaluation in solid tumors is mainly complicated by the diagnostic yield of standard imaging modalities and tumor size contradictions. Exploring clinical/molecular features that complement the use of radiologic and bronchoscopy findings may provide significant values for a more accurate pathologic determination of nodal metastasis. Methods: We analyzed surgery-resected tumor samples from 72 treatment-naïve lung adenocarcinoma (LUAD) patients via whole-exome sequencing (WES). Mutational profiles of patients categorized based on their pathologic lymph node metastasis (pN) status were assessed, and the association between metastatic-driving features and overall survival (OS) was then explored in both the study cohort and the Cancer Genome Atlas Program (TCGA) external cohort. Results: Among all 72 patients, 41 (56.9%) were pathologically confirmed as positive for lymph node metastasis (pN-positive), while 31 (43.1%) were categorized as pN-negative. The most frequently mutated gene was TP53 in both subgroups (54.8% and 53.7%, respectively), and the aberrant KEAP1-NRF2 signaling pathway was found significantly more enriched in pN-negative patients. Copy-number variant analysis showed that chromosome 7p amplifications (X7p_amp) at the arm level and EGFR amplifications at the focal gene level were more frequently identified in pN-positive patients (P=0.008 and P=0.004, respectively). Importantly, these amplifications were associated with worse OS in both the study cohort and the TCGA external dataset. Consistent with the unfavorable prognosis, pN-positive patients demonstrated a higher chromosome instability score (CIS) (median: 0.32 vs. 0.14, P=0.14) and a lower tumor mutation burden (TMB) (median: 1.83 vs. 3.85 muts/Mb, P=0.048) compared to pN-negative patients. Conclusion: Our findings suggest that X7p_amp, particularly EGFR amplification, might be associated with pathologic involvement of lymph nodes and an unfavorable prognosis in LUAD patients. Our study highlights the importance of integrating clinical and genomic data into conventional radiologic modalities for a more accurate determination of pathologic nodal involvement. Citation Format: Shijie Wang, Zhiwei Zhou, Song Wang, Rongyun Guo, Zeming Ma, Dachuan Zhao, Liang Wang, Yinan Liu, Yuanyuan Ma, Jianzhi Zhang, Sha Wang, Yedan Chen, Haimeng Tang, Qiuxiang Ou, Jinfeng Chen. Identifying genomic features associated with pathologic lymph node metastasis in lung adenocarcinoma patients [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 5047.

Abstract 1638: Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis

Abstract The preservation of spindle pole integrity is crucial for proper spindle assembly and chromosome segregation in mitosis, yet the precise mechanisms governing spindle pole integrity remain elusive. During mitosis, phosphorylated Ensa (p-ENSA) localizes to spindle poles, and the inhibition of ENSA leads to a range of mitotic defects, including misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome aberrations, resulting in a delayed mitotic progression. Remarkably, the mitotic delay induced by ENSA inhibition is alleviated upon depletion of PP2A-B55α, but spindle pole defects persist. Significantly, we have observed an interaction between ENSA and Aurora A during mitosis, and the inhibition of ENSA diminishes Aurora A expression at the mitotic spindle poles. Intriguingly, the injection of MKI-2-sensitized tumors is associated with heightened chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. These findings offer new insights into the regulation of spindle pole integrity through the MASTL-ENSA-Aurora A pathway during mitosis, emphasizing the pivotal role of ENSA in recruiting Aurora A to the spindle pole, independently of PP2A-B55α. Citation Format: Seul Kim, Kyoungho Jun, Ye-Hyun Kim, Jae Sung Kim. Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis [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 1638.

Abstract 322: Ultraplex genome and exome sequencing of rare single cells

Abstract Single-cell genome analysis enables researchers to gain novel insights into tumor heterogeneity. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is limited. PCR-based amplification methods normally have high error rates, low coverage uniformity, and extensive allelic drop-outs making interpretation of SNVs and CNVs challenging.Here we introduce a new whole-genome amplification method which utilizes an optimized isothermal DNA amplification reaction with a Hi-fidelity, error correcting polymerase and downstream library preparation protocol to allow pooling of amplified DNA. The key DNA amplification steps use multiple displacement amplification (MDA) technology to amplify gDNA directly from single cells, nuclei or previously isolated genomic DNA. During the MDA reaction, sample-barcodes are incorporated into the newly synthetized DNA, allowing the amplified DNA to be pooled and processed in to one ultraplex library. This method saves library preparation time, reduces DNA library costs and limits plastic consumable usage.Our current workflow allows 24 samples to be multiplexed in one library and 24 libraries to be produced for a total of 576 samples per sequencing flow cell. This method can be used to investigate chromosomal instability with low pass sequencing or SNV and CNV analysis following whole genome, exome-capture workflows. Citation Format: Ioanna Andreou, Samuel Rulli, Katja Heitz, Silke Huebner, Christin Meerschiff, Eric Lader. Ultraplex genome and exome sequencing of rare single cells [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 322.

Abstract 5723: Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay

Abstract Kinesin superfamily proteins (KIFs) are a large family of molecular motor proteins, that share a highly conserved motor domain. KIFs play an important role in cell division and transport of vesicles and organelles within cells. Mitotic kinesins hydrolyze ATP through its ATPase activity to move along the spindle microtubules and carry out several functions during mitosis to facilitate precise chromosome segregation. Altered expressions of several kinesins are shown in various cancers, fueling the aggressive nature of cancers leading to genomic instability. KIF11/Eg5 overexpression is shown in various cancers including hepatic carcinoma, lung, prostate, colorectal, gastric, and pancreatic cancers. Small molecule inhibitors of KIF11/Eg5 and CENP-E are shown to result in mitotic arrest leading to apoptosis in tumor cell lines. Despite the poor clinical outcome of several Eg5 and CENP-E inhibitors entered in clinical trials, targeting kinesins remains an attractive anti-cancer therapeutic approach. Recently, an inhibitor of KIF18A has entered clinical trial for treatment of solid tumors with high chromosomal instability. Here we show development of a microplate based biochemical screening assays for a panel of kinesin motor domain proteins. We summarize kinetic characterization of kinesin proteins and study the selectivity of various previously reported KIF18A, Eg5, CENP-E inhibitors across a kinesin selectivity panel. Citation Format: Brenna P. Lee, Sung Won Oh, Kurumi Y. Horiuchi, Safnas F. AbdulSalam. Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay [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 5723.

Intratumoral TREX1 induction promotes immune evasion by limiting type I interferon.

Chromosomal instability is a hallmark of human cancer that is associated with aggressive disease characteristics. Chromosome mis-segregations help fuel natural selection, but they risk provoking a cGAS-STING immune response through the accumulation of cytosolic DNA. The mechanisms of how tumors benefit from chromosomal instability while mitigating associated risks, such as enhanced immune surveillance, are poorly understood. Here, we identify cGAS-STING-dependent upregulation of the nuclease TREX1 as an adaptive, negative feedback mechanism that promotes immune evasion through digestion of cytosolic DNA. TREX1 loss diminishes tumor growth, prolongs survival of host animals, increases tumor immune infiltration, and potentiates response to immune checkpoint blockade selectively in tumors capable of mounting a type I interferon response downstream of STING. Together, these data demonstrate that TREX1 induction shields chromosomally unstable tumors from immune surveillance by dampening type I interferon production and suggest that TREX1 inhibitors might be used to selectively target tumors that have retained the inherent ability to mount an interferon response downstream of STING.

Allele-specific transcriptional effects of subclonal copy number alterations enable genotype-phenotype mapping in cancer cells

Subclonal copy number alterations are a prevalent feature in tumors with high chromosomal instability and result in heterogeneous cancer cell populations with distinct phenotypes. However, the extent to which subclonal copy number alterations contribute to clone-specific phenotypes remains poorly understood. We develop TreeAlign, which computationally integrates independently sampled single-cell DNA and RNA sequencing data from the same cell population. TreeAlign accurately encodes dosage effects from subclonal copy number alterations, the impact of allelic imbalance on allele-specific transcription, and obviates the need to define genotypic clones from a phylogeny a priori, leading to highly granular definitions of clones with distinct expression programs. These improvements enable clone-clone gene expression comparisons with higher resolution and identification of expression programs that are genomically independent. Our approach sets the stage for dissecting the relative contribution of fixed genomic alterations and dynamic epigenetic processes on gene expression programs in cancer.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques

BackgroundNaturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers.MethodsWe conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation.ResultsSimilar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs.ConclusionsThese data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.Graphical abstract

BUB1/KIF14 complex promotes anaplastic thyroid carcinoma progression by inducing chromosome instability.

Chromosome instability (CIN) is a common contributor driving the formation and progression of anaplastic thyroid cancer (ATC), but its mechanism remains unclear. The BUB1 mitotic checkpoint serine/threonine kinase (BUB1) is responsible for the alignment of mitotic chromosomes, which has not been thoroughly studied in ATC. Our research demonstrated that BUB1 was remarkably upregulated and closely related to worse progression-free survival. Knockdown of BUB1 attenuated cell viability, invasion, migration and induced cell cycle arrests, whereas overexpression of BUB1 promoted the cell cycle progression of papillary thyroid cancer cells. BUB1 knockdown remarkably repressed tumour growth and tumour formation of nude mice with ATC xenografts and suppressed tumour metastasis in a zebrafish xenograft model. Inhibition of BUB1 by its inhibitor BAY-1816032 also exhibited considerable anti-tumour activity. Further studies showed that enforced expression of BUB1 evoked CIN in ATC cells. BUB1 induced CIN through phosphorylation of KIF14 at serine1292 (Ser1292 ). Overexpression of the KIF14ΔSer1292 mutant was unable to facilitate the aggressiveness of ATC cells when compared with that of the wild type. Collectively, these findings demonstrate that the BUB1/KIF14 complex drives the aggressiveness of ATC by inducing CIN.

Prolonged Particulate Hexavalent Chromium Exposure Induces DNA Double-Strand Breaks and Inhibits Homologous Recombination Repair in Primary Rodent Lung Cells.

Hexavalent chromium [Cr(VI)] is a known lung carcinogen and a driving mechanism in human lung cells for Cr(VI)-induced lung cancer is chromosome instability, caused by prolonged Cr(VI) exposure inducing DNA double-strand breaks, while simultaneously inhibiting the repair of these breaks. In North Atlantic right whales, Cr(VI) induces breaks but does not inhibit repair. It is unclear if this repair inhibition is specific to human lung cells or occurs in other species, as it has only been considered in humans and North Atlantic right whales. We evaluated these outcomes in rodent cells, as rodents are an experimental model for metal-induced lung carcinogenesis. We used a guinea pig lung fibroblast cell line, JH4 Clone 1, and rat lung fibroblasts. Cells were exposed to two different particulate Cr(VI) compounds, ranging from 0 to 0.5 ug/cm2, for 24 or 120h and assessed for cytotoxicity, DNA double-strand breaks, and DNA double-strand break repair. Both particulate Cr(VI) compounds induced a concentration-dependent increase in cytotoxicity and DNA double-strand breaks after acute and prolonged exposures. Notably, while the repair of Cr(VI)-induced DNA double-strand breaks increased after acute exposure, the repair of these breaks was inhibited after prolonged exposure. These results are consistent with outcomes in human lung cells indicating rodent cells respond like human cells, while whale cells have a markedly different response.

Kinesin-7 CENP-E in tumorigenesis: Chromosome instability, spindle assembly checkpoint, and applications.

Kinesin motors are a large family of molecular motors that walk along microtubules to fulfill many roles in intracellular transport, microtubule organization, and chromosome alignment. Kinesin-7 CENP-E (Centromere protein E) is a chromosome scaffold-associated protein that is located in the corona layer of centromeres, which participates in kinetochore-microtubule attachment, chromosome alignment, and spindle assembly checkpoint. Over the past 3decades, CENP-E has attracted great interest as a promising new mitotic target for cancer therapy and drug development. In this review, we describe expression patterns of CENP-E in multiple tumors and highlight the functions of CENP-E in cancer cell proliferation. We summarize recent advances in structural domains, roles, and functions of CENP-E in cell division. Notably, we describe the dual functions of CENP-E in inhibiting and promoting tumorigenesis. We summarize the mechanisms by which CENP-E affects tumorigenesis through chromosome instability and spindle assembly checkpoints. Finally, we overview and summarize the CENP-E-specific inhibitors, mechanisms of drug resistances and their applications.

Prognostic and predictive role of immune microenvironment in colorectal cancer.

Colorectal cancer (CRC) represents a molecularly heterogeneous disease and one of the most frequent causes of cancer-related death worldwide. The traditional classification of CRC is based on pathomorphological and molecular characteristics of tumor cells (mucinous, ring-cell carcinomas, etc.), analysis of mechanisms of carcinogenesis involved (chromosomal instability, microsatellite instability, CpG island methylator phenotype) and mutational statuses of commonly altered genes (KRAS, NRAS, BRAF, APC, etc.), as well as expression signatures (CMS 1-4). It is also suggested that the tumor microenvironment is a key player in tumor progression and metastasis in CRC. According to the latest data, the immune microenvironment can also be predictive of the response to immune checkpoint inhibitors. In this review, we highlight how the immune environment influences CRC prognosis and sensitivity to systemic therapy.

TP63–TRIM29 axis regulates enhancer methylation and chromosomal instability in prostate cancer

BackgroundProstate adenocarcinoma (PRAD) is the second leading cause of cancer-related deaths in men. High variability in DNA methylation and a high rate of large genomic rearrangements are often observed in PRAD.ResultsTo investigate the reasons for such high variance, we integrated DNA methylation, RNA-seq, and copy number alterations datasets from The Cancer Genome Atlas (TCGA), focusing on PRAD, and employed weighted gene co-expression network analysis (WGCNA). Our results show that only single cluster of co-expressed genes is associated with genomic and epigenomic instability. Within this cluster, TP63 and TRIM29 are key transcription regulators and are downregulated in PRAD. We discovered that TP63 regulates the level of enhancer methylation in prostate basal epithelial cells. TRIM29 forms a complex with TP63 and together regulates the expression of genes specific to the prostate basal epithelium. In addition, TRIM29 binds DNA repair proteins and prevents the formation of the TMPRSS2:ERG gene fusion typically observed in PRAD.ConclusionOur study demonstrates that TRIM29 and TP63 are important regulators in maintaining the identity of the basal epithelium under physiological conditions. Furthermore, we uncover the role of TRIM29 in PRAD development.

Limited contribution of photoenzymatic DNA repair in mitigating carry-over effects from larval UVB exposure: Implications for frog recruitment

Solar ultraviolet-B (UVB) radiation has increased due to stratospheric ozone depletion, climate and ecosystem changes and is a driver of amphibian population declines. Photoenzymatic repair (PER) is a critical mechanism for limiting UVB lethality in amphibian larvae. However, the link between PER and the UVB-induced effects remains understudied through long-term investigations in vivo. Here, we assessed how larval PER determines the lethal and sublethal effects induced by environmentally relevant acute UVB exposure until the juvenile phase in the Neotropical frog Odontophrynus americanus. We conducted laboratory-based controlled experiments in which tadpoles were or were not exposed to UVB and subsequently were exposed to light (for PER activation) or dark treatments. Results showed that the rates of mortality and apoptosis observed in post-UVB dark treatment are effectively limited in post-UVB light treatment, indicating PER (and not dark repair, i.e. nucleotide excision repair) is critical to limit the immediate genotoxic impact of UVB-induced pyrimidine dimers. Nonetheless, even tadpoles that survived UVB exposure using PER showed sublethal complications that extended to the juvenile phase. Tadpole responses included alterations in morphology, chromosomal instability, increased skin susceptibility to fungal proliferation, as well as increased generation of reactive oxygen species. The short-term effects were carried over to later stages of life because metamorphosis time increased and juveniles were smaller. No body abnormalities were visualized in tadpoles, metamorphs, and juveniles, suggesting that O. americanus is UVB-resistant concerning these responses. This study reveals that even frog species equipped with an effective PER are not immune to carry-over effects from early UVB exposure, which are of great ecological relevance as late metamorphosis and smaller juveniles may impact individual performance and adult recruitment to breeding. Future ecological risk assessments and conservation and management efforts for amphibian species should exercise caution when linking PER effectiveness to UVB resistance.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

AurkA/TPX2 co-overexpression in nontransformed cells promotes genome instability through induction of chromosome mis-segregation and attenuation of the p53 signalling pathway

The Aurora-A kinase (AurkA) and its major regulator TPX2 (Targeting Protein for Xklp2) are key mitotic players frequently co-overexpressed in human cancers, and the link between deregulation of the AurkA/TPX2 complex and tumourigenesis is actively investigated. Chromosomal instability, one of the hallmarks of cancer related to the development of intra-tumour heterogeneity, metastasis and chemo-resistance, has been frequently associated with TPX2-overexpressing tumours. In this study we aimed to investigate the actual contribution to chromosomal instability of deregulating the AurkA/TPX2 complex, by overexpressing it in nontransformed hTERT RPE-1 cells. Our results show that overexpression of both AurkA and TPX2 results in increased AurkA activation and severe mitotic defects, compared to AurkA overexpression alone. We also show that AurkA/TPX2 co-overexpression yields increased aneuploidy in daughter cells and the generation of micronucleated cells. Interestingly, the p53/p21 axis response is impaired in AurkA/TPX2 overexpressing cells subjected to different stimuli; consistently, cells acquire increased ability to proliferate after independent induction of mitotic errors, i.e. following nocodazole treatment. Based on our observation that increased levels of the AurkA/TPX2 complex affect chromosome segregation fidelity and interfere with the activation of a pivotal surveillance mechanism in response to altered cell division, we propose that co-overexpression of AurkA and TPX2 per se represents a condition promoting the generation of a genetically unstable context in nontransformed human cells.

Transcriptome analysis of polyploid giant cancer cells and their progeny reveals a functional role for p21 in polyploidization and depolyploidization

Polyploid giant cancer cells (PGCC) are frequently detected in tumors and are increasingly recognized for their roles in chromosomal instability and associated genome evolution that leads to cancer recurrence. We previously reported that therapy stress promotes polyploidy, and that acid ceramidase plays a role in depolyploidization. In this study, we used an RNA-seq approach to gain a better understanding of the underlying transcriptomic changes that occur as cancer cells progress through polyploidization and depolyploidization. Our results revealed gene signatures that are associated with disease-free and/or overall survival in several cancers and identified the cell cycle inhibitor CDKN1A/p21 as the major hub in PGCC and early progeny. Increased expression of p21 in PGCC was limited to the cytoplasm. We previously demonstrated that the sphingolipid enzyme acid ceramidase is dispensable for polyploidization upon therapy stress but plays a crucial role in depolyploidization. The current study demonstrates that treatment of cells with ceramide is not sufficient for p53-independent induction of p21 and that knockdown of acid ceramidase, which hydrolyzes ceramide, does not interfere with upregulation of p21. In contrast, blocking the expression of p21 with UC2288 prevented the induction of acid ceramidase and inhibited both the formation of PGCC from parental cells as well as the generation of progeny from PGCC. Taken together, our data suggest that p21 functions upstream of acid ceramidase and plays an important role in polyploidization and depolyploidization.

Abstract B033: Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability

Abstract Diffuse gliomas are pediatric high-grade gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. Previously we have reported that chromosome missegregation triggers H3.3 S31 phosphorylation along chromosome arms in ana/telophase leading to p53-induced cell cycle arrest. Here we show, in vitro and in vivo, that H3.3 K27M, G34R, and G34V suppress S31 phosphorylation by its mitotic kinase, Chk1. In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used CRISPR/Cas9 gene editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of glioma. Expression of PDGFß linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDFß-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single glioma-associated point mutation in H3.3 can generate chromosomal instability, and which is a major contributing factor to H3.3 K27M and G34-altered glioma formation. Citation Format: Charles A. Day, Florina Grigore, Alyssa Langfald, Faruck Hakkim, David Daniels, Kevin Vaughan, James Robinson, Edward Hinchcliffe. Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B033.

The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Integrated chromosomal instability and tumor microbiome redefined prognosis-related subtypes of pancreatic cancer

BackgroundPancreatic cancer is a common malignancy with poor prognosis and limited treatment. Here we aimed to investigate the role of host chromosomal instability (CIN) and tumor microbiome in the prognosis of pancreatic cancer patients. MethodsOne hundred formalin-fixed paraffin-embedded (FFPE) pancreatic cancer samples were collected. DNA extracted from FFPE samples were analyzed by low-coverage whole-genome sequencing (WGS) via a customized bioinformatics workflow named ultrasensitive chromosomal aneuploidy detector. ResultsSamples are tested according to the procedure of ultrasensitive chromosomal aneuploidy detector (UCAD). We excluded 2 samples with failed quality control, 1 patient lost to follow-up and 6 dead in the perioperative period. The final 91 patients were admitted for the following analyses. Thirteen (14.3%) patients with higher CIN score had worse overall survival (OS) than those with lower CIN score. The top 20 microbes in pancreatic cancer samples included 15 species of bacteria and 5 species of viruses. Patients with high human herpesvirus (HHV)-7 and HHV-5 DNA reads exhibited worse OS. Furthermore, we classified 91 patients into 3 subtypes. Patients with higher CIN score (n =13) had the worst prognosis (median OS 6.9 mon); patients with lower CIN score but with HHV-7/5 DNA load (n = 24) had worse prognosis (median OS 10.6 mon); while patients with lower CIN score and HHV-7/5 DNA negative (n = 54) had the best prognosis (median OS 21.1 mon). ConclusionsHigh CIN and HHV-7/5 DNA load were associated with worse survival of pancreatic cancer. The novel molecular subtypes of pancreatic cancer based on CIN and microbiome had prognostic value.

A genome-wide study of gastric intramucosal neoplasia based on somatic copy number alterations, gene mutations, and mRNA expression patterns.

We performed comprehensive analyses of somatic copy number alterations (SCNAs) and gene expression profiles of gastric intramucosal neoplasia (IMN) using array-based methods in 97 intestinal-type IMNs, including 39 low-grade dysplasias (LGDs), 37 high-grade dysplasias (HGDs), and 26 intramucosal carcinomas (IMCs) with stromal invasion of the lamina propria to identify the molecular mechanism of IMN. In addition, we examined gene mutations using gene panel analyses. We used cluster analyses for exclusion of arbitrariness to identify SCNA patterns and expression profiles. IMNs were classified into two distinct subgroups (subgroups 1 and 2) based on SCNA patterns. Subgroup 1 showed a genomic stable pattern due to the low frequency of SCNAs, whereas subgroup 2 exhibited a chromosomal instability pattern due to the high frequencies of SCNAs and TP53 mutations. Interestingly, although the frequencies of LGD and HGD were significantly higher in subgroup 1 than in subgroup 2, IMC was commonly found in both types. Although the expression profiles of specific mRNAs could be used to categorise subgroups 1 and 2, no clinicopathological findings correlated with either subgroup. We examined signalling pathways specific to subgroups 1 and 2 to identify the association of each subgroup with signalling pathways based on gene ontology tree visualisation: subgroups 1 and 2 were associated with haem metabolism and chromosomal instability, respectively. These findings reveal a comprehensive genomic landscape that highlights the molecular complexity of IMNs and provide a road map to facilitate our understanding of gastric IMNs.