Minute Chromosomes Research Articles

A standardized protocol for sample preparation for scanning electron microscopy to visualize extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) are circular DNA structures associated with cancer and drug resistance. One specific type, double minute (DM) chromosomes, has been studied since the 1960s using imaging techniques like cytogenetics and fluorescence microscopy. Specialized techniques such as atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer micro to nano-scale visualization, but current sample preparation methods may not optimally preserve ecDNA structure. Our study introduces a systematic protocol using SEM for high-resolution ecDNA visualization. We have optimized the end-to-end procedure, providing a standardized approach to explore the circular architecture of ecDNA and address the urgent need for better understanding in cancer research.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [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 6957.

Abstract 7348: CytoCellDB: A gold standard database for classification and analysis of extrachromosomal DNA in cancer

Abstract Extrachromosomal DNA (ecDNA), or double minute chromosomes, are established cytogenetic markers for malignancy and genome instability. More recently, the cancer community has gained a heightened awareness of the roles of ecDNA in cancer proliferation, drug resistance and epigenetic remodeling. A current hindrance to understanding the biological roles of ecDNA is the lack of available cell line model systems with experimental cytogenetic data that confirm ecDNA status. Although several recent landmark studies have identified numerous cell lines and tumor models with ecDNA, the current sample size limits our ability to detect ecDNA-driven molecular differences due to limitations in power. Increasing the number of model systems that are known to express ecDNA would provide new avenues for understanding the fundamental underpinnings of ecDNA biology and would unlock a wealth of potential targeting strategies for ecDNA-driven cancers. To bridge this gap, we created CytoCellDB, a resource that provides karyotype annotations, including ecDNA status, and leverages publicly available global cell line data from the Cancer Dependency Map (DepMap) and the Cancer Cell Line Encyclopedia (CCLE). We expanded the total number of cancer cell lines with ecDNA annotations to 632, or 34% of the cell lines in CCLE/DepMap. Of these, 154 cell lines express ecDNA, which is a 240% increase from the current sample size. We demonstrate that a strength of our framework is the ability to interrogate ecDNA, and the compendium of other chromosomal aberrations, in the context of cancer-specific vulnerabilities, drug sensitivities, and other multi-omic features (genomics, transcriptomics, methylation, proteomics). We anticipate that this resource will lower the barrier to systematic, population-scale discoveries related to ecDNA and will provide insights into strategies and best practices for determining novel therapeutics that overcome ecDNA-mediated drug resistance. Citation Format: Liz Brunk. CytoCellDB: A gold standard database for classification and analysis of extrachromosomal DNA in cancer [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 7348.

Optical Genome Mapping for Comprehensive Cytogenetic Analysis of Soft-Tissue and Bone Tumors for Diagnostic Purposes

Soft tissue and bone tumours represent a heterogeneous group of tumours encompassing more than 100 histological subtypes today. Identifying genetic aberrations is increasingly important in these tumours for accurate diagnosis. While gene mutations are typically detected by second generation sequencing, the identification of structural variants (SVs) and copy number alterations (CNAs) remains challenging and requires various cytogenetic techniques including karyotyping, fluorescent in situ hybridisation and arrays, each having important limitations.We applied Optical Genome Mapping (OGM), a non-sequencing-based technique for high-resolution detection of SVs and CNAs in a retrospective series of diagnostic soft tissue and bone tumour samples. Sample preparation was successful in 38 out of 53 cases, with the highest success rate in non-adipocytic soft tissue tumours (24/27 cases, 89%). In 32 out of 35 cases carrying a diagnostic SV or CNA, OGM identified the aberration (91%) including a POU2AF3::EWSR1 fusion in a round cell sarcoma and a translocation t(1;5)(p22;p15) in a myxoinflammatory fibroblastic sarcoma. Interestingly, OGM shed light on the genomic complexity underlying the various aberrations. In five samples OGM showed that chains of rearrangements generated the diagnostic fusion, three of which involved chromoplexy. Additionally, in nine samples chromothripsis was causal to the formation of giant marker/ring/double minute chromosomes. Finally, compared to standard-of-care cytogenetics, OGM revealed additional aberrations, requiring further investigation of their potential clinical relevance.

Advancements in Focal Amplification Detection in Tumor/Liquid Biopsies and Emerging Clinical Applications.

Focal amplifications (FAs) are crucial in cancer research due to their significant diagnostic, prognostic, and therapeutic implications. FAs manifest in various forms, such as episomes, double minute chromosomes, and homogeneously staining regions, arising through different mechanisms and mainly contributing to cancer cell heterogeneity, the leading cause of drug resistance in therapy. Numerous wet-lab, mainly FISH, PCR-based assays, next-generation sequencing, and bioinformatics approaches have been set up to detect FAs, unravel the internal structure of amplicons, assess their chromatin compaction status, and investigate the transcriptional landscape associated with their occurrence in cancer cells. Most of them are tailored for tumor samples, even at the single-cell level. Conversely, very limited approaches have been set up to detect FAs in liquid biopsies. This evidence suggests the need to improve these non-invasive investigations for early tumor detection, monitoring disease progression, and evaluating treatment response. Despite the potential therapeutic implications of FAs, such as, for example, the use of HER2-specific compounds for patients with ERBB2 amplification, challenges remain, including developing selective and effective FA-targeting agents and understanding the molecular mechanisms underlying FA maintenance and replication. This review details a state-of-the-art of FA investigation, with a particular focus on liquid biopsies and single-cell approaches in tumor samples, emphasizing their potential to revolutionize the future diagnosis, prognosis, and treatment of cancer patients.

Double minute chromosomes in Anastrepha fraterculus (Wiedemann) (DIPTERA: Tephritidae): a model for cancer studies

Anastrepha fraterculus (Wied.) is known as the South American Fruit Fly. In order to develop efficient control strategies, we need deep knowledge on its biology along with periodical studies on population dynamics. Citogenetic studies on natural populations of this pest fly made it possible to reveal the presence of double minute chromosomes (DMs) in several natural populations of this insect. Cytogenetic studies performed in our insect laboratory, allowed the genetic analysis through a genealogical methodology. The establishment of families made it possible the genetic studies allowing the rigorous identification, characterization and confirmation of new chromosomal variants, frequently missinterpreted when the materials analyzed only come from nature. Double minute chromosomes -considered a type of chromosomal rearrangement- are extra chromosomal gene copies. This study sought to answer: What role do DMs chromosomes play in laboratory populations of the pest derived from natural populations? How are they transmitted from one generation to another? To answer our questions, we analyzed for 25 generations, two laboratory populations derived from Tucuman (T) and Buenos Aires (BA -tester) guava,. Data were recovered from cytological analysis of ganglia preparations revealed with H33258. The natural population from BA didn’t carry DMs. Results showed DMs in flies of both laboratory populations. DMs were transmitted from parents to progenies through 25 generations and their transmission was randomized in number. DMs are the cytological expression of resistance mechanisms used by the pest as a response to environmental stress. DMs in BA strain marked the change to the laboratory environment. A. fraterculus is a model insect for the study of cancer.

Methotrexate-resistance is associated with double minute chromosomes in lymphocytes from patients with prostate cancer

Human lymphocytes have long been used to assess the gene damage occurring in vivo as well as in vitro . In this study , peripheral blood lymphocytes from patients with prostate cancer were cultivated in vitro short-term culture in the presence of methotrexate (MTX) to investigate the possible mechanism of resistance to MTX in those cells. The results indicate that the resistance to MTX in lymphocytes from prostate cancer patients is associated with dihydrofolate reductase (DHFR) gene amplification . Metaphase chromosome preparations from those cells were examined and were shown to contain double minute chromosomes (DMs). We conclude, that the presence of DMs may support that the resistance to MTX in prostate cancer patients may be associated with DHFR gene amplification.

Clinical and Histological Features of Ovarian Hypoplasia/Dysgenesis in Alpacas.

Alpacas have a high incidence of congenital reproductive tract abnormalities, including ovarian hypoplasia/dysgenesis. Diagnosis of this condition is often challenging. The present study describes the clinical, ultrasonographic, and histologic features of ovarian hypoplasia/dysgenesis syndrome in 5 female alpacas. Additionally, serum AMH levels were compared between female alpacas diagnosed with ovarian hypoplasia/dysgenesis and a group of reproductively sound females (n = 11). The syndrome was suspected based on the presence of an infantile uterus and lack of ovaries by ultrasonography and laparoscopy. All females had normal female karyotype (n = 74 XX), but one presented a minute chromosome. The ovaries from these cases showed 3 main histological classifications: hypoplasia (n = 2), dysgenesis (n = 2), and dysplasia (n = 1). Serum AMH levels in affected females were significantly lower (P < 0.05) than those of reproductively sound control females. In conclusion, Serum AMH level may be helpful in the rapid diagnosis of ovarian hypoplasia/dysgenesis syndrome in alpacas. Furthermore, this syndrome in alpacas presents a variety of histological features. Different mechanisms may be involved in the derangement of ovarian differentiation. Further studies are needed to elucidate the causes of the syndrome.

HolistIC: leveraging Hi-C and whole genome shotgun sequencing for double minute chromosome discovery.

Double minute (DM) chromosomes are acentric extrachromosomal DNA artifacts that are frequently observed in the cells of numerous cancers. They are highly amplified and contain oncogenes and drug-resistance genes, making their presence a challenge for effective cancer treatment. Algorithmic discovery of DM can potentially improve bench-derived therapies for cancer treatment. A hindrance to this task is that DMs evolve, yielding circular chromatin that shares segments from progenitor DMs. This creates DMs with overlapping amplicon coordinates. Existing DM discovery algorithms use whole genome shotgun sequencing (WGS) in isolation, which can potentially incorrectly classify DMs that share overlapping coordinates. In this study, we describe an algorithm called 'HolistIC' that can predict DMs in tumor genomes by integrating WGS and Hi-C sequencing data. The consolidation of these sources of information resolves ambiguity in DM amplicon prediction that exists in DM prediction with WGS data used in isolation. We implemented and tested our algorithm on the tandem Hi-C and WGS datasets of three cancer datasets and a simulated dataset. Results on the cancer datasets demonstrated HolistIC's ability to predict DMs from Hi-C and WGS data in tandem. The results on the simulated data showed the HolistIC can accurately distinguish DMs that have overlapping amplicon coordinates, an advance over methods that predict extrachromosomal amplification using WGS data in isolation. Our software, named 'HolistIC', is available at http://www.github.com/mhayes20/HolistIC. Supplementary data are available at Bioinformatics online.

Double minute chromosomes in acute myeloid leukemia and myelodysplastic syndromes are associated with complex karyotype, monosomal karyotype, TP53 deletion, and TP53 mutations

Double minute chromosomes (DMs) are rare in hematologic malignancies. We presented the cytogenetic characteristics and clinical features of the largest single-center cohort of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients with DMs. A total of 2576 AML patients and 1642 MDS patients were investigated, and 30 patients (AML = 19; MDS = 11) who had DMs were followed up. DMs were more common in primary AML (94.7%) and MDS (90.9%). Monosomal karyotypes (MK) were also the main cytogenetic characteristics, like complex karyotypes (CK). AML with myelodysplasia-related changes (AML-MRC) and MDS-refractory anemia with excess blasts (MDS-RAEB) was common in this cohort. We conclude that DMs-positive AML and DMs-positive MDS are associated with older age, complex karyotypes, monosomal karyotypes, TP53 deletion and TP53 mutations. DMs are a type of chromothripsis, which can be observed by the karyotype analysis. MYC and KMT2A were the most commonly amplified genes in DMs. Most patients with DMs presented an extremely poor prognosis.

Nonrandom Chromosomal Abnormalities in Acute Nonlymphocytic Leukemia in Patients Treated for Hodgkin Disease and Non-Hodgkin Lymphomas

Chromosomal analyses of myeloid cells were performed on ten patients who had acute nonlymphocytic leukemia (ANLL) following treatment for malignant lymphoma. Seven patients had Hodgkin disease and three had non-Hodgkin lymphoma, poorly differentiated lymphocytic type. Six patients were treated with radiotherapy and chemotherapy; two had radiotherapy only, and two chemotherapy only. The median time between diagnosis of lymphoma and subsequent leukemia was 58 mo. Four patients had the blast phase of a myeloproliferative syndrome, four had acute myelogenous leukemia, one had acute promyelocytic leukemia, and the tenth, erythroleukemia. None of four patients whose leukemia was treated with intensive chemotherapy responded. Every patient had an abnormal karyotype. Seven of the patients showed hypodiploid cell lines, two a pseudodiploid, and one a hyperdiploid cell line. Cells from every patient except one were lacking a B chromosome; in eight, this could be identified as a No. 5. Five of nine patients were lacking a No. 7. Loss or rearrangement of No. 17 was found in four and of Nos. 6 or 8 in three patients. Many of the karyotypes were bizarre, with marker chromosomes and minute chromosomes. The karyotypic pattern seen in these patients showed no correlation with the nature of the originalmore » lymphoma, the type of leukemia, or the therapy used. The chromosomal pattern of hypodiploid cell lines found in ANLL that arose de novo was similar to that occurring in treated lymphoma. However, in ANLL de novo, less than half of the patients had fewer than 46 chromosomes, and less than 10% had fewer than 45 chromosomes. In this study, 70% of the patients had fewer than 46 and 40% had fewer than 45 chromosomes. The critical question thus concerns the factors, as yet unknown, that predispose to the development of hypodiploid modal numbers in ANLL in lymphoma.« less

HolistIC: Leveraging Hi-C and Whole Genome Shotgun Sequencing for Double Minute Chromosome Discovery

Abstract Double minute chromosomes are acentric extrachromosomal DNA artifacts that are frequently observed in the cells of numerous cancers. They are highly amplified and contain oncogenes and drug resistance genes, making their presence a challenge for effective cancer treatment. Algorithmic discovery of double minutes (DM) can potentially improve bench-derived therapies for cancer treatment. A hindrance to this task is that DMs evolve, yielding circular chromatin that shares segments from progenitor double minutes. This creates double minutes with overlapping amplicon coordinates. Existing DM discovery algorithms use whole genome shotgun sequencing in isolation, which can potentially incorrectly classify DMs that share overlapping coordinates. In this study, we describe an algorithm called “ HolistIC” that can predict double minutes in tumor genomes by integrating whole genome shotgun sequencing (WGS) and Hi-C sequencing data. The consolidation of these sources of information resolves ambiguity in double minute amplicon prediction that exists in DM prediction with WGS data used in isolation. We implemented and tested our algorithm on the tandem Hi-C and WGS datasets of three cancer datasets and a simulated dataset. Results on the cancer datasets demonstrated HolistIC’s ability to predict DMs from Hi-C and WGS data in tandem. The results on the simulated data showed the HolistIC can accurately distinguish double minutes that have overlapping amplicon coordinates, an advance over methods that predict extrachromosomal amplification using WGS data in isolation. Availability Our software is available at http://www.github.com/mhayes20/HolistIC.

MYC amplification on double minute chromosomes in plasma cell leukemia with double IGH/CCND1 fusion genes

In multiple myeloma (MM), MYC rearrangements that result in increased MYC expression are associated with an aggressive form of MM and adverse outcome. However, the consequences of MYC amplification in MM remain unclear. Here, we describe an unusual case of plasma cell leukemia (PCL) harboring MYC amplification on double minute chromosomes (dmin). A 79-year-old woman was initially diagnosed as having BJP-κ type MM with bone lesions. After seven months, the disease progressed to secondary PCL: leukocytes 49.1 × 109/L with 77% plasma cells showing lymphoplasmacytic appearance. The bone marrow was infiltrated with 76% plasma cells immunophenotypically positive for CD38 and negative for CD45, CD19, CD20, and CD56. The karyotype by G-banding and spectral karyotyping was 48,XX,der(14)t(11;14)(q13;q32),+der(14)t(14;19)(q32;q13.1),+18,6~95dmin[15]/46,XX[5]. Fluorescence in situ hybridization detected multiple MYC signals on dmin and double IGH/CCND1 fusion signals on der(14)t(11;14) and der(14)t(14;19). Most plasma cells were diffusely and strongly positive for MYC and CCND1 by immunohistochemistry. The patient died of progressive disease after one week. MYC amplification led to high expression of MYC and rapid disease progression, indicating its clinical significance in the pathogenesis of MM/PCL. MYC amplification on dmin may be a very rare genetic event closely associated with the progression to PCL and coexistence of IGH/CCND1 fusions.

A hidden Markov model-based approach to reconstructing double minute chromosome amplicons

Double minute chromosomes (DMs) are circular fragments of extrachromosomal DNA. They cause extreme gene amplification in the cells of malignant tumours. Their existence correlates with malignant tumour cell behaviour and drug resistance. Locating DMs is important for informing precision therapy to cancer treatment. Furthermore, accurate detection of double minutes requires precise reconstruction of their amplicons, which are the highly-amplified gene-carrying contiguous segments that adjoin to form DMs. This work presents AmpliconFinder - a Hidden-Markov Model-based approach to detect DM amplicons. To assess its efficacy, AmpliconFinder was used to augment an earlier framework for DM detection (DMFinder), thus improving its sensitivity and robustness to noisy sequence data. Experiments on simulated genomic data show that augmenting DMFinder with AmpliconFinder significantly increased the sensitivity of DMFinder on these data. Moreover, DMFinder with AmpliconFinder found all previously reported DMs in three pediatric medulloblastoma datasets, whereas the original DMFinder framework found none.

A hidden Markov model-based approach to reconstructing double minute chromosome amplicons

A hidden Markov model-based approach to reconstructing double minute chromosome amplicons

Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.

The cytoskeletal interacting protein Septin 9 (SEPT9), a member of the septin gene family, has been proposed to have oncogenic functions. It is a known hot spot of retroviral tagging insertion and a fusion partner of both de novo and therapy-induced mixed lineage leukemia (MLL). Of all septins, SEPT9 holds the strongest link to cancer, especially breast cancer. Murine models of breast cancer frequently exhibit Sept9 amplification in the form of double minute chromosomes, and about 20% of human breast cancer display genomic amplification and protein over expression at the SEPT9 locus. Yet, a clear mechanism by which SEPT9 elicits tumor-promoting functions is lacking.To obtain unbiased insights on molecular signatures of SEPT9 upregulation in breast tumors, we overexpressed several of its isoforms in breast cancer cell lines. Global transcriptomic profiling supports a role of SEPT9 in invasion. Functional studies reveal that SEPT9 upregulation is sufficient to increase degradation of the extracellular matrix, while SEPT9 downregulation inhibits this process. The degradation pattern is peripheral and associated with focal adhesions (FA), where it is coupled with increased expression of matrix metalloproteinases. SEPT9 overexpression induces MMP upregulation in human tumors and in culture models and promotes MMP3 secretion to the media at FAs. Downregulation of SEPT9 or chemical inhibition of septin filament assembly impairs recruitment of MMP3 to FAs. Our results indicate that SEPT9 promotes upregulation and both trafficking and secretion of MMPs near FAs, thus enhancing migration and invasion of breast cancer cells.

Chromosome-Level Alpaca Reference Genome VicPac3.1 Improves Genomic Insight Into the Biology of New World Camelids.

The development of high-quality chromosomally assigned reference genomes constitutes a key feature for understanding genome architecture of a species and is critical for the discovery of the genetic blueprints of traits of biological significance. South American camelids serve people in extreme environments and are important fiber and companion animals worldwide. Despite this, the alpaca reference genome lags far behind those available for other domestic species. Here we produced a chromosome-level improved reference assembly for the alpaca genome using the DNA of the same female Huacaya alpaca as in previous assemblies. We generated 190X Illumina short-read, 8X Pacific Biosciences long-read and 60X Dovetail Chicago® chromatin interaction scaffolding data for the assembly, used testis and skin RNAseq data for annotation, and cytogenetic map data for chromosomal assignments. The new assembly VicPac3.1 contains 90% of the alpaca genome in just 103 scaffolds and 76% of all scaffolds are mapped to the 36 pairs of the alpaca autosomes and the X chromosome. Preliminary annotation of the assembly predicted 22,462 coding genes and 29,337 isoforms. Comparative analysis of selected regions of the alpaca genome, such as the major histocompatibility complex (MHC), the region involved in the Minute Chromosome Syndrome (MCS) and candidate genes for high-altitude adaptations, reveal unique features of the alpaca genome. The alpaca reference genome VicPac3.1 presents a significant improvement in completeness, contiguity and accuracy over VicPac2 and is an important tool for the advancement of genomics research in all New World camelids.

An open label, multicenter, phase II study of KRT-232, an oral small molecule inhibitor of MDM2, for the treatment of patients with Merkel cell carcinoma (MCC) who have failed treatment with anti-PD-1/L1 immunotherapy.

TPS9602 Background: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a poor prognosis in patients with advanced disease, with mortality as high as 46%. An oncogenic Merkel cell polyomavirus (MCPyV) is present in about 80% of MCC tumors, which inactivates tumor suppressors p53 and RB. Virus-positive MCC has proven to be an immunologically responsive disease and the recently approved PD-L1 inhibitor avelumab (March 2017) has improved the prognosis of patients with advanced MCC. However, despite the addition of immune checkpoint inhibitors to the treatment paradigm, a lack of response or disease progression occurs in up to two-thirds of avelumab patients previously treated with chemotherapy, and 32-44% of first-line patients treated with the PD-1 inhibitors nivolumab or pembrolizumab. KRT-232 is a potent and selective small molecule targeted drug that binds to murine double minute chromosome 2 (MDM2) and inhibits the MDM2/tumor protein 53 (p53) protein-protein interaction. Inactivating mutations in p53 have rarely been found in MCC, and the majority of MCCs with the presence of MCPyV express p53WT. The purpose of this study is to evaluate the tolerability and efficacy of KRT-232 in p53WT MCC patients who have been treated with anti-PD-1/L1 immunotherapy. Methods: The primary objective is ORR by RECIST v1.1 after 2 cycles (42 days) as determined by independent review. A Simon two-stage design is employed. In Stage 1, up to 13 eligible patients will be enrolled and treated with 240 mg KRT-232 once daily (QD) on Days 1-7 of a 21-day cycle. If the predesignated efficacy threshold is reached in Stage 1, enrollment for Stage 2 will include an additional 14 patients (for a total of 27 patients). The study is enrolling in the U.S. Clinical trial information: NCT03787602.

Structure and evolution of double minutes in diagnosis and relapse brain tumors

Double minute chromosomes are extrachromosomal circular DNA fragments frequently found in brain tumors. To understand their evolution, we characterized the double minutes in paired diagnosis and relapse tumors from a pediatric high-grade glioma and four adult glioblastoma patients. We determined the full structures of the major double minutes using a novel approach combining multiple types of supporting genomic evidence. Among the double minutes identified in the pediatric patient, only one carrying EGFR was maintained at high abundance in both samples, whereas two others were present in only trace amounts at diagnosis but abundant at relapse, and the rest were found either in the relapse sample only or in the diagnosis sample only. For the EGFR-carrying double minutes, we found a secondary somatic deletion in all copies at relapse, after erlotinib treatment. However, the somatic mutation was present at very low frequency at diagnosis, suggesting potential resistance to the EGFR inhibitor. This mutation caused an in-frame RNA transcript to skip exon 16, a novel transcript isoform absent in EST database, as well as about 700 RNA-seq of normal brains that we reviewed. We observed similar patterns involving longitudinal copy number shift of double minutes in another four pairs (diagnosis/relapse) of adult glioblastoma. Overall, in three of five paired tumor samples, we found that although the same oncogenes were amplified at diagnosis and relapse, they were amplified on different double minutes. Our results suggest that double minutes readily evolve, increasing tumor heterogeneity rapidly. Understanding patterns of double minute evolution can shed light on future therapeutic solutions to brain tumors carrying such variants.

Sei-1 promotes double minute chromosomes formation through activation of the PI3K/Akt/BRCA1-Abraxas pathway and induces double-strand breaks in NIH-3T3 fibroblasts

Sei-1 is a potential oncogene that plays an important role in promoting genomic instability. Double minute chromosomes (DMs) are hallmarks of gene amplification and contribute to tumorigenesis. Defects in the DNA double-strand break (DSB) repairing pathways can lead to gene amplification. To date, the mechanisms governing the formation of DMs induced by Sei-1 are not fully understood. We established DMs induced by Sei-1 in the NIH-3T3 cell line. RNA-sequencing was used to identify key characteristics of differentially expressed genes. Metaphase spreads were used to calculate DM numbers. Immunofluorescence was employed to detect γH2AX foci. Western blot and Akt pathway inhibition experiments were performed to reveal the role of the PI3K/Akt/BRCA1-Abraxas pathway in Sei-1-induced DMs. Luciferase reporter assay was employed to explore the regulatory mechanisms between Sei-1 and BRCA1. DM formation was associated with a deficiency in DSB repair. Based on this finding, activation of the PI3K/Akt/BRCA1-Abraxas pathway was found to increase the DM population with passage in vivo, and inhibition resulted in a reduction of DMs. Apart from this, it was shown for the first time that Sei-1 could directly regulate the expression of BRCA1. Our results suggest that the PI3K/Akt/BRCA1-Abraxas pathway is responsible for the formation of DMs induced by Sei-1.