Homogeneously Staining Regions Research Articles

Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Abstract B092: Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy

Abstract Background: Focal high copy number oncogene amplification frequently occurs on extrachromosomal DNA (ecDNA), acentric circular DNA derived from chromosomes during neoplastic transformation and tumor progression. The unique properties of ecDNA facilitate a high degree of genomic plasticity and tumor heterogeneity, which enables rapid acquired resistance to targeted therapies and results in poor patient outcomes. Reintegrated ecDNA, also referred to as homogeneously staining regions (HSR), can exist in dynamic equilibrium with their circular form, although their potential adaptive and resistance properties have not been well characterized. Elucidating the genomic plasticity and mechanisms afforded to cancer cells harboring HSR may provide insights into new therapeutic approaches necessary to address this ecDNA-derived focal amplification. Methods: A novel model was developed that recapitulates the genomic plasticity of focal oncogene amplifications involving FGFR2 and EGFR in response to the FGFR inhibitor, infigratinib. Results: KATOIII gastric cancer cells harbor focal amplification of the FGFR2 oncogene (absolute copy number (CN) = 136 based on next-generation sequencing (NGS) that resides on an HSR as determined by FISH. Short- and long-read NGS revealed prior DNA breakpoints consistent with circular structures containing FGFR2, suggestive of reintegrated ecDNA as HSR. Despite initial sensitivity to FGFR2 inhibition (EC50 = 20 nM), cells acquired resistance following infigratinib dose escalation within 12 weeks. Infigratinib-resistant KATOIII cells retained the FGFR2 HSR while further amplifying FGFR2 on ecDNA, which correlated with increased FGFR2 CN, mRNA, and protein expression. Structural breakpoint analysis of the FGFR2 amplicon revealed conservation between baseline and ecDNA-amplified infigratinib resistant cells, suggesting that the HSR gave rise to ecDNA. Removal of infigratinib resulted in near-complete disappearance of FGFR2 ecDNA and return to near-baseline FGFR2 CN, protein expression, and signaling within 3 weeks. Strikingly, at high infigratinib concentrations that effectively inhibit FGFR2 signaling, cells rapidly switched to EGFR amplified on ecDNA, with concomitant reduction of FGFR2 ecDNA (CN = 553 to 238) and signaling and increased EGFR (CN = 3 to 30), mRNA, and EGFR signaling. Following ecDNA switching from FGFR2 to EGFR, KATOIII cells remained insensitive to infigratinib while acquiring sensitivity to the EGFR inhibitor erlotinib. Mutational analysis revealed no new oncogenic mutations including FGFR2 and EGFR, indicating the EGFR overexpression afforded by ecDNA was sufficient to drive acquired resistance to high infigratinib concentrations. Conclusions: These findings indicate that tumor cells with ecDNA-derived chromosomally reintegrated HSR retain the capacity to amplify ecDNA, thus enabling rapid adaptation to therapeutic pressure. Consequently, new treatment strategies that disable adaptive genomic plasticity are needed to address oncogene amplified cancers that leverage ecDNA biology. Citation Format: Kristen Turner, Homa Hemmati, Cory DuPai, John Bibay, Claire Klebs, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Intrinsic genomic plasticity of extrachromosomal DNA (ecDNA) enables oncogene amplified tumor cells to develop rapid acquired resistance to targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B092.

ATACAmp: a tool for detecting ecDNA/HSRs from bulk and single-cell ATAC-seq data

BackgroundHigh oncogene expression in cancer cells is a major cause of rapid tumor progression and drug resistance. Recent cancer genome research has shown that oncogenes as well as regulatory elements can be amplified in the form of extrachromosomal DNA (ecDNA) or subsequently integrated into chromosomes as homogeneously staining regions (HSRs). These genome-level variants lead to the overexpression of the corresponding oncogenes, resulting in poor prognosis. Most existing detection methods identify ecDNA using whole genome sequencing (WGS) data. However, these techniques usually detect many false positive regions owing to chromosomal DNA interference.ResultsIn the present study, an algorithm called “ATACAmp” that can identify ecDNA/HSRs in tumor genomes using ATAC-seq data has been described. High chromatin accessibility, one of the characteristics of ecDNA, makes ATAC-seq naturally enriched in ecDNA and reduces chromosomal DNA interference. The algorithm was validated using ATAC-seq data from cell lines that have been experimentally determined to contain ecDNA regions. ATACAmp accurately identified the majority of validated ecDNA regions. AmpliconArchitect, the widely used ecDNA detecting tool, was used to detect ecDNA regions based on the WGS data of the same cell lines. Additionally, the Circle-finder software, another tool that utilizes ATAC-seq data, was assessed. The results showed that ATACAmp exhibited higher accuracy than AmpliconArchitect and Circle-finder. Moreover, ATACAmp supported the analysis of single-cell ATAC-seq data, which linked ecDNA to specific cells.ConclusionsATACAmp, written in Python, is freely available on GitHub under the MIT license: https://github.com/chsmiss/ATAC-amp. Using ATAC-seq data, ATACAmp offers a novel analytical approach that is distinct from the conventional use of WGS data. Thus, this method has the potential to reduce the cost and technical complexity associated ecDNA analysis.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate‑resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double‑strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)‑resistant HT29 cells [DM‑ and homogeneously staining region (HSR)‑containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM‑containing cells. No corresponding changes were found in HSR‑containing cells. The present study further verified the involvement of MSH3‑regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3‑depleted DM‑containing cells. Finally, changes in MSH3 expression could affect DHFR amplification‑derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA‑based drug resistance in cancer.

Establishment and characteristics of GWH04, a new primary human glioblastoma cell line.

Glioblastoma multiforme (GBM) is a common and fatal disease of the central nervous system. GBM cell lines are fundamental tools used in GBM research. The establishment of novel continuous GBM cell lines with clear genetic backgrounds could facilitate the exploration of molecular mechanisms and the screening and evaluation of antitumor drugs in GBM studies. In the present study, a novel primary glioblastoma cell line was established, named GWH04, from a patient with GBM, and its STR genotype and various tumor parameters were examined. The STR information of GWH04 was identical to that of the original primary tumor tissue. Compared with existing cell lines, GWH04 had a similar in vitro proliferation rate as the U87 cell line, but a faster rate than the GL15 cell line, and substantial soft agar clone-formation capacity and subcutaneous and intracranial tumorigenic capacity. For drug sensitivity test, half maximal inhibitory concentration assays for multiple drugs were performed in these three cell lines, and GWH04 cells were found to be resistant to temozolomide. Aneuploid karyotype with a median of 84 chromosomes was possessed by GWH04, as well as chromosomal structural abnormalities, such as broken chromosomes, double centromere chromosomes, homogeneous staining regions, and double microbodies. Gene sequencing further revealed the mutational status of genes TP53, PTEN, PDGFRA, ERBB2, BRCA1, NF1, and MLH1 and the promoter region of telomerase reverse transcriptase (C228T) in this cell line. Altogether, these results indicated that GWH04 will be a useful tool for human GBM studies both in vitro and in vivo.

Karyotypic analysis and isolation of four DNA markers of the scleractinian coral Favitespentagona (Esper, 1795) (Scleractinia, Anthozoa, Cnidaria).

We performed conventional and molecular cytogenetic studies on the Favitespentagona Esper, 1795, a scleractinian coral mostly found along the west coast of Japan. Karyotype analysis of F.pentagona by G-banding revealed a karyogram containing a homogenously staining region (HSR) on chromosome 10 in more than 50% of the examined metaphase spreads. This HSR consisted of sequences from 18S ribosomal RNA (rRNA) genes, as demonstrated by fluorescence in situ hybridization (FISH) and DNA sequencing. We highlighted the development of four chromosomal FISH markers from repetitive genes such as U2 small nuclear RNA linked to 5S rRNA sequence (U2 snRNA-5S), 18S rRNA, histone H3, and uncharacterized gene FP-9X. The chromosomal locations of the U2 snRNA-5S and 18S RNA were on the terminal end of long arm of chromosomes 2 and 10, respectively, while the histone H3 and the uncharacterized gene were located near the centromeres of chromosomes 1 and 9, respectively. These FISH markers will improve the karyotyping of F.pentagona from mitotic preparations which helps in widening our understanding of coral genetic structure and chromosome organization. In addition, these improvements in karyotyping will provide the basis in constructing of chromosome-level genome assembly for F.pentagona.

The Significance of Chromosome 11 Abnormalities in Patients with AML

Introduction: At the time of diagnosis AML the finding of KMT2A gene rearrangement qualifies patient to the group with adverse cytogenetic risk according to ELN 2017. In many hematooncological diseases including AML abnormalities concerning another regions of chromosome 11 are detected. However, their significance for outcome, CR rate, OS is still unknown. The main aim of this study was analysis of chromosome 11 abnormalities in AML patients, focusing on these without KMT2A gene rearrangement and its influence on patients outcome.Methods: Karyotype analysis from bone marrow aspirate was performed for patients with chromosome 11 abnormalities (abn11, n=36) and compared to two groups: with complex (CK, n=20) and normal karyotype (NK, n=44). The basic clinical and laboratory data including cytogenetic (karyotype and FISH method) and presence of TP53 deletion were analyzed to characterize in detail the group of patients with chromosome 11 abnormalities.Patients were treated with intensive chemotherapy, in 33% of them allogeneic cell transplantation (alloHCT) was performed.Results: In patients with abn11 the most frequent type of aberration was translocation, 11q23(+)/KMT2A(-) and 11q10-25 as a main region involved in aberrations, with minimal narrowing of region to 11q22. In patients with abn11 and translocation (excluding t(9;11)) there was higher frequency occurrence of 1 to 2 additional abnormalities (p=0.033) and lower frequency of complex karyotype existence (p=0.026). The gain of genetic material from chromosome 11 was observed in 33% of patients with abn11 group and the minimal amplified region was 11q22. Furthermore, in patients with amplification of chromosome 11 region, different additional cytogenetic abnormalities (such as monosomy 5 (p=0.045) and 18 (p<0.001)) were found in comparison to patients with other abnormalities of chromosome 11. Additional KMT2Agene copies were often observed in patients with 11q10-25 (p=0.013) and 11pter-qter (p=0.022) region involvement. The lowest frequency of TP53 deletion was in NK group (p=0.002) but comparable in patients with CK and abn11 without impact on OS and CR. The FISH analysis did not revealed KMT2A gene rearrangement in group with normal karyotype. There was particular attention to 11q23 region in patients with AML which enabled abnormality detection in GTG analysis, furthermore KMT2A rearrangement was confirmed or excluded by FISH method. In few cases FISH analysis allowed verification of KMT2A gene status in case of atypical hybrydization pattern occurrence.Leukocyte counts (p<0.001) and type of AML (de novo, secondary, therapy-related, p=0.022) differs in patients with abn11 in comparison to remained studied subgroups (Table 1.). The lowest blast percentage was observed in patients with 11p rearrangement (p=0.045). There were no significant differences in OS between all 3 groups of patients. CR was achieved in 15% in a CK group and it was significantly lower than in NK and abn11 group (p<0.001). Furthermore alloHCT improve OS in all treated patients (p<0.001). Additional genetic material from chromosome 11 occurrence had negative impact on OS (p<0.001) and CR (p=0.007). Patients with abn11 who achieved CR had lower median age (p=0.01) compared to these without CR. The lowest percentage of CR in abn11 group was observed in patients with additional KMT2A gene copies (p=0.008), 11q10-25 region involvement (p=0.036), HSR (homogeneously staining region) and aneuploidy (p=0.04).Conclusions: In summary, the most frequent region involved in chromosome 11 aberration was 11q22. Additional material derived from chromosome 11 had negative impact on CR and OS. Cytogenetic and molecular analysis in patients with AML are needed to detect most of abn11. In the era of targeted therapies their identification is very important to qualify the patient for more effective treatment. Both cytogenetic and molecular techniques widespread knowledge about cancer biology, however, classic cytogenetic is still “gold standard” and crucial method in AML diagnosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gene Amplification and the Extrachromosomal Circular DNA.

Oncogene amplification is closely linked to the pathogenesis of a broad spectrum of human malignant tumors. The amplified genes localize either to the extrachromosomal circular DNA, which has been referred to as cytogenetically visible double minutes (DMs), or submicroscopic episome, or to the chromosomal homogeneously staining region (HSR). The extrachromosomal circle from a chromosome arm can initiate gene amplification, resulting in the formation of DMs or HSR, if it had a sequence element required for replication initiation (the replication initiation region/matrix attachment region; the IR/MAR), under a genetic background that permits gene amplification. In this article, the nature, intracellular behavior, generation, and contribution to cancer genome plasticity of such extrachromosomal circles are summarized and discussed by reviewing recent articles on these topics. Such studies are critical in the understanding and treating human cancer, and also for the production of recombinant proteins such as biopharmaceuticals by increasing the recombinant genes in the cells.

Speckled 100kDa gene in pigs: Alternative splicing, subcellular localization, and response to interferon-α stimulation.

Speckled 100kDa (Sp100) plays an important role in the antiviral immune response, however, little is known about porcine Sp100. In this study, porcine Sp100 was cloned and its response to interferon (IFN) α was identified. We obtained the cDNA (V1) of the gene, SP100, and seven alternative splicing variants (V2-8). Isoform V1 encoded a 386 amino acid protein and contained a homogeneously-staining region (HSR) domain. Isoforms V3, 4, 6 and 7 were deletion/insertion variants and contained HSR domain as V1. The splicing of porcine SP100 was very complicated and many transcripts existed as revealed by cloning and minigene analyses. Using GFP-fusion constructs isoforms V1, 3, 4, 6 and 7 were localized to nucleus and the nuclear localization signal was identified as PSNRKRR at positions 331-337 of V1. Porcine SP100 was unevenly distributed in all tissues studied and differentially expressed between pigs with different disease-resistance/susceptibilities. Porcine SP100 was strongly increased by IFNα due to the existence of an IFN-stimulated response element in the promoter. A single nucleotide-70A>C polymorphism enhanced promoter activity. The results provided the basis for determining the role of Sp100 in antiviral responses and may assist in breeding pigs with high disease-resistance.

Homogeneously Staining Regions (HSR) in Chromosome 1 of the House Mouse: Synapsis and Recombination at Meiosis

Amplified sequences constitute a large part of mammalian genomes. A chromosome 1 containing 2 large (up to 50 Mb) homogeneously staining regions (HSRs) separated by a small inverted euchromatic region is present in many natural populations of the house mouse (Mus musculus musculus). The HSRs are composed of a long-range repeat cluster, Sp100-rs, with a repeat length of 100 kb. In order to understand the organization and function of HSRs in meiotic chromosomes, we examined synapsis and recombination in male mice hetero- and homozygous for the HSR-carrying chromosome using FISH with an HSR-specific DNA probe and immunolocalization of the key meiotic proteins. In all homozygous and heterozygous pachytene nuclei, we observed fully synapsed linear homomorphic bivalents 1 marked by the HSR FISH probe. The synaptic adjustment in the heterozygotes was bilateral: the HSR-carrying homolog was shortened and the wild-type homolog was elongated. The adjustment was reversible: desynapsis at diplotene was accompanied by elongation of the HSRs. Immunolocalization of H3K9me2/3 indicated that the HSRs in the meiotic chromosome retained the epigenetic modification typical for C-heterochromatin in somatic cells. MLH1 foci, marking mature recombination nodules, were detected in the proximal HSR band in heterozygotes and in both HSR bands of homozygotes. Unequal crossing over within the long-range repeat cluster can cause variation in size of the HSRs, which has been detected in the natural populations of the house mouse.

Molecular structure and evolution mechanism of two populations of double minutes in human colorectal cancer cells.

Gene amplification chiefly manifests as homogeneously stained regions (HSRs) or double minutes (DMs) in cytogenetically and extrachromosomal DNA (ecDNA) in molecular genetics. Evidence suggests that gene amplification is becoming a hotspot for cancer research, which may be a new treatment strategy for cancer. DMs usually carry oncogenes or chemoresistant genes that are associated with cancer progression, occurrence and prognosis. Defining the molecular structure of DMs will facilitate understanding of the molecular mechanism of tumorigenesis. In this study, we re‐identified the origin and integral sequence of DMs in human colorectal adenocarcinoma cell line NCI‐H716 by genetic mapping and sequencing strategy, employing high‐resolution array‐based comparative genomic hybridization, high‐throughput sequencing, multiplex‐fluorescence in situ hybridization and chromosome walking techniques. We identified two distinct populations of DMs in NCI‐H716, confirming their heterogeneity in cancer cells, and managed to construct their molecular structure, which were not investigated before. Research evidence of amplicons distribution in two different populations of DMs suggested that a multi‐step evolutionary model could fit the module of DM genesis better in NCI‐H716 cell line. In conclusion, our data implicated that DMs play a very important role in cancer progression and further investigation is necessary to uncover the role of the DMs.

Double-strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation.

Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low-dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low-dose hydroxyurea also induces random double-strand breakage throughout the nucleus. In the present study, we found that double-strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double-strand breakage in cancer cells. Because double-strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.

High Level MYC Amplification in Aggressive B-Cell Lymphomas: Is It a Marker of Aggressive Disease?

MYC amplification (amp) is a marker of poor prognosis in many non-hematologic malignancies. While MYC translocations in B cell lymphoma (BCL) have been extensively studied, little is known about the significance of MYC amp. Recent studies describe increased MYC copy numbers (3-10 copies/cell) to be associated with more aggressive BCL. The WHO 2017 does not include MYC amp in the definition of high-grade BCL (HGBCL) with MYC and BCL2 and/or BCL6 rearrangement (“double-hit lymphoma”, DHL). However, it also states that high-level MYC amp occurring together with a MYC rearrangement, and concurrent with BCL2 rearrangement likely has a similar clinical impact as classic DHL. Although increased MYC copy number is commonly identified by routine fluorescence in situ hybridization (FISH) testing in BCL, the experience of our large cytogenetics reference laboratory suggests that high-level MYC amp, defined as uncountable MYC signals, is far less common. Therefore, we sought to characterize the clinical, pathologic and cytogenetic features of patients with BCL showing high-level MYC amp.The Mayo Clinic cytogenetic database was retrospectively reviewed for all cases of BCL with high-level MYC amp seen by FISH from January 2010 - February 2018. All FISH studies reported as MYC amp were re-reviewed by a cytogeneticist to verify the level of amp and the MYC probes involved. Pathology was reviewed by two independent hematopathologists. Clinical information was collected through chart review. Survival analysis was performed using Kaplan-Meier curves and the Wilcoxon rank-sum test.FISH analysis for MYC aberrations identified 44/9715 (0.45%) cases with high-level MYC amp. Of cases with available H&E, the most common morphology was diffuse large BCL (DLBCL) (82%; 28/34), followed by HGBCL (15%; 5/34) and plasmablastic BCL (3%; 1/34). Hans cell of origin (COO) algorithm immunohistochemistry (IHC) identified 21/25 (84%) germinal center B-cell-like (GCB), and 4/25 (16%) non-GCB cases. 21/27 (78%) cases were BCL2+ by IHC. MYC+ by IHC was ≥40% in 21/28 (75%) and <40% in 7/28 (25%) cases. 9/17 (53%) were “double expressers” (DEL) by IHC.MYC amp probe signals appeared in a cloud-like distribution (CLD) in 31 (70%) or in a single homogenous staining region (HSR) in 13 (30%) [Figure 1A]. Among 38 cases with amp in a MYC break-apart probe, 21 (55%) had amp of 5‘ alone, 15 (40%) of intact and 2 (5%) of the 3‘ probe alone. 7/44 (16%) had MYC translocations (5 IGH; 2 non-IGH). BCL2 rearrangement was seen in 15/39 (38%) cases, and BCL6 rearrangement in 3/36 (8%). Only 2/44 (4%) cases met the current WHO 2017 definition of DHL.Clinical data was available for 20 cases. Median age at diagnosis was 64.5 (range 25-88) years with M:F of 1.5:1. Only 1/14 (7%) had bone marrow while 16/18 (88.8%) had other extranodal sites of involvement (8 gastrointestinal). The clinical presentation was heterogeneous: 13 de novo, 3 post-transplant, 3 transformed from low grade and 1 mediastinal BCL. 9/14 had an elevated LDH, median 387 (225 - 2063) U/L. R-CHOP was the most common first line therapy in 12/17 (70%). At median follow-up of 18.2 (range 0.4 - 88.9) months, 9 patients had died, 3 were in relapse and 8 remained in first complete remission. Lymphoma relapse/progression was the most common cause of death in 7/9 (78%). The median overall survival (OS) was 29.3 months [Figure 1B]. There was no statistically significant difference in OS by morphologic classification (DLBCL vs HGBCL, p=0.6), double expresser (Yes/No, p=0.19), COO (GCB vs non-GCB, p=0.08), MYC amp pattern (HSR vs CLD, p=0.48), MYC amp probe (3‘ vs 5‘ vs intact, p=0.31), MYC rearrangement (Yes/No, p=0.1), or MYC amp with concurrent BCL2/BCL6 rearrangement (Yes/No ,p=0.2).To our knowledge, this is the first study to characterize the clinical, pathologic and cytogenetic features of BCL with strictly-defined high level MYC amp identified by FISH. The 5' signal alone, or the intact MYC probe are most frequently amplified, and two distinct patterns of amp can be seen. Predominant extra nodal involvement is an important clinical observation. These cases are usually DLBCL, GCB type, and infrequently have concurrent MYC/BCL2/BCL6 rearrangement. Our study suggests that BCL with high-level MYC amp may have an aggressive disease course regardless of MYC, BCL2 and BCL6 gene rearrangement status. A larger series is necessary to further understand the clinical significance of high-level MYC amp in BCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Establishment and characterization of a novel vincristine-resistant diffuse large B-cell lymphoma cell line containing the 8q24 homogeneously staining region.

Chromosome band 8q24 is the most frequently amplified locus in various types of cancers. MYC has been identified as the primary oncogene at the 8q24 locus, whereas a long noncoding gene, PVT1, which lies adjacent to MYC, has recently emerged as another potential oncogenic regulator at this position. In this study, we established and characterized a novel cell line, AMU‐ML2, from a patient with diffuse large B‐cell lymphoma (DLBCL), displaying homogeneously staining regions at the 8q24 locus. Fluorescence in situ hybridization clearly detected an elevation in MYC copy numbers corresponding to the homogenously staining region. In addition, a comparative genomic hybridization analysis using high‐resolution arrays revealed that the 8q24 amplicon size was 1.4 Mb, containing the entire MYC and PVT1 regions. We also demonstrated a loss of heterozygosity for TP53 at 17p13 in conjunction with a TP53 frameshift mutation. Notably, AMU‐ML2 cells exhibited resistance to vincristine, and cell proliferation was markedly inhibited by MYC‐shRNA‐mediated knockdown. Furthermore, genes involved in cyclin D, mTOR, and Ras signaling were downregulated following MYC knockdown, suggesting that MYC expression was closely associated with tumor cell growth. In conclusion, AMU‐ML2 cells are uniquely characterized by homogenously staining regions at the 8q24 locus, thus providing useful insights into the pathogenesis of DLBCL with 8q24 abnormalities.

Amplification of a transgene within a long array of replication origins favors higher gene expression in animal cells.

Plasmids with both a mammalian replication initiation region (IR) and a matrix attachment region (MAR) are spontaneously amplified in transfected cells, and generate extrachromosomal double minute (DM) or chromosomal homogeneously staining region (HSR). We previously isolated the shortest core IR (G5) required for gene amplification. In this study, we ligated the G5 DNA to create direct or inverted repeats, mixed the repeats with an expression plasmid, and transfected the mixture into human COLO 320DM or hamster CHO DG44 cells. Consequently, we found that the transfected sequence generated DMs or HSR where, surprisingly, the plasmid sequence was embedded within a long stretch of G5 sequences. The amplified structure from the direct G5 repeats was stable, whereas that from the inverted repeats was not. The amplification might be explained by the efficient replication/multimerization of the G5 repeat and recombination with the co-transfected plasmid in an extrachromosomal context. The product might then be integrated into a chromosome arm to generate a HSR. The expression from the plasmid within the long G5 array was much higher than that from a simple plasmid repeat. Because G5 is a core IR that favors gene expression, a long array of G5 provides an excellent environment for gene expression from the embedded plasmid.

Establishment of a Novel DLBCL Cell Line: AMU-ML2, Derived from a Primary Refractory Patient Shows Homogeneous Staining Region of 8q24 Inducing High Expression of Long Non-Coding RNAs Encoded By PVT1 and Resistance to Vincristine

Introduction: Primary refractory DLBCL is an extremely difficult condition to treat and represent an unmet medical need. The clarification of the molecular pathogenesis of this disease can contribute the development of new therapeutic possibilities. PVT1 is located adjacent to MYC at 8q24 and a non-protein coding gene. PVT1 produces a variety of long non-coding RNAs (lncRNAs) and has now emerged as a potential regulator in the pathogenesis of a lot of cancers. However, the precise biological and clinical significance of PVT1 remains largely unknown. In this study we established a novel human DLBCL cell line with hsr (homogeneous staining region) of 8q24 inducing high expression of PVT1 lncRNAs, named AMU-ML2. The cell line was established from a patient with primary refractory DLBCL before initiation of chemotherapy. We analyzed the genetic characteristics and investigated the drug sensitivity of the cell line in comparison with other B-cell lymphoma cell lines which had 8q24 abnormalities.Case: A 64-year-old man was diagnosed as DLBCL involved with bone marrow and pleural effusion. He was refractory to initial R-CHOP and subsequent R-hyper-CVAD/MA therapy and died of Trichosporon asahiisepsis 6 months after diagnosis. His lymphoma cells at diagnosis were positive for CD19, CD20, BCL6 and HLA-DR, and negative for CD3, CD5, CD10, cyclinD1, BCL2, MUM1, TP53 and EBER by flow cytometry and/or immunohistochemical staining, indicative of germinal center B-cell-like (GCB) DLBCL. The representative karyotype of cells was 46,XY, including add(6)(p11), add(8)(q24), hsr 8q24, add(9)(p13) and add(17)(p11.2).Establishment of the cell line: After 2 weeks of culture, the cells in pleural effusion collected before chemotherapy started to grow in suspension. The cell line was designated as AMU-ML2 after confirmation that the cells started growing again after the conventional freeze-thaw procedure.Materials and methods: B-cell lymphoma cell lines; AMU-ML2, SU-DHL-10, Raji, P3HR-1 and VAL were used in the present study. These cell lines had t(8;14)(q24;q32) or 8q24 amplification. The genomic aberration of AMU-ML2 was analyzed by cytogenetics including G-banding and FISH (fluorescence in situ hybridization) combined with array-CGH (SurePrint G3, Agilent). The TaqMan real time RT-PCR was used for measurement of expression levels of MYC and PVT1. MTT assay was used for the cell proliferation to analyze the drug sensitivities of cyclophosphamide, doxorubicin, vincristine (VCR) and prednisolone.Results: AMU-ML2 cells showed same immunophenotypic feature and karyotype as the primary sample from the patient. FISH using a MYC/IGH probe set showed no fusion signal for IGH and MYC; however, the MYC copy number was extremely increased, corresponding to hsr on chromosome 8q24. Array-CGH revealed that a 1,462 kb region, containing both the entire MYC and PVT1 genes at 8q24.21, was amplified, with greater than 20 copies present in cells (Figure 1). In addition to amplification of the MYC/PVT1 region at 8q24, 14 additional copy number alterations were detected. These included segment losses on 6p22.1-6p21.31 and 17p13 led to the LOH of human leukocyte antigen (HLA) loci and TP53, respectively (Figure 1). Using the real time RT-PCR, the expression level of the PVT1 lncRNAs were highest in AMU-ML2 among other B-cell lymphoma cell lines that we used, while the expression level of MYC in AMU-ML2 was relatively low. The proliferation rate of AMU-ML2 was significantly higher after 72-h exposure to VCR (100, 500 and 1,000 nM) compared with other cell lines (Figure 2).Discussion: AMU-ML2 was established in 2-week culture from a refractory patient before starting the chemotherapy, therefore this cell line seems to reflect the real nature of primary refractory DLBCL, not related to chemotherapy and/or cell culture. MYC amplification and the LOH of TP53 and HLA may contribute to the development of lymphoma in our case. High expression of PVT1 lncRNAs was likely to be more related with drug resistance to VCR than MYC expression, although AMU-ML2 had co-amplification of MYC and PVT1. Thus, AMU-ML2 can provide insight into the genetic and biological features and the therapeutic approaches in primary refractory DLBCL. [Display omitted] [Display omitted] DisclosuresUeda:Mundipharma KK: Consultancy; Kyowa Hakko Kirin: Research Funding.

Epigenetic Repeat-Induced Gene Silencing in the Chromosomal and Extrachromosomal Contexts in Human Cells.

A plasmid bearing both a replication initiation region and a matrix attachment region is spontaneously amplified in transfected mammalian cells and generates plasmid repeats in the extrachromosomal double minutes (DMs) or the chromosomal homogeneously staining region (HSR). Generally, the repeat sequences are subject to repeat-induced gene silencing, the mechanism of which remains to be elucidated. Previous research showed that gene expression from the same plasmid repeat was higher from repeats located at DMs than at the HSR, which may reflect the extrachromosomal environment of the DMs. In the current study, plasmid repeats in both DMs and HSR were associated with repressive histone modifications (H3K9me3, H3K9me2), and the levels of repressive chromatin markers were higher in HSR than in DMs. Inactive chromatin is known to spread to neighboring regions in chromosome arm. Here, we found that such spreading also occurs in extrachromosomal DMs. Higher levels of active histone modifications (H3K9Ac, H3K4me3, and H3K79me2) were detected at plasmid repeats in DMs than in HSR. The level of DNA CpG methylation was generally low in both DMs and HSR; however, there were some hypermethylated copies within the population of repeated sequences, and the frequency of such copies was higher in DMs than in HSR. Together, these data suggest a “DNA methylation-core and chromatin-spread” model for repeat-induced gene silencing. The unique histone modifications at the extrachromosomal context are discussed with regard to the model.

Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells

INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity.

Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing.

Plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) are spontaneously amplified in transfected mammalian cells, and such amplification generates chromosomal homogeneously staining regions (HSRs) or extrachromosomal double minutes (DMs). This method provides a novel, efficient, and rapid way to establish cells that stably produce high levels of recombinant proteins. However, because IR/MAR plasmids are amplified as repeats, they are frequently targeted by repeat-induced gene silencing (RIGS), which silences a variety of repeated sequences in transgenes and the genome. To address this problem, we developed a novel screening system using the IR/MAR plasmid to isolate human genome sequences that alleviate RIGS. The screen identified a 3,271 bp sequence (B-3-31) that elevated transgene expression without affecting the amplification process. Neither non-B structure (i.e., the inverted repeats or bending) nor known epigenetic modifier elements such as MARs, insulators, UCOEs, or STARs could explain the anti-silencing activity of B-3-31. Instead, the activity was distributed throughout the entire B-3-31 sequence, which was extremely A/T-rich and CpG-poor. Because B-3-31 effectively and reproducibly alleviated RIGS of repeated genes, it could be used to increase recombinant protein production.

Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells

BackgroundGene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs)...