Zelda Lichtensztejn

3D Telomere FISH defines LMP1-expressing Reed–Sternberg cells as end-stage cells with telomere-poor ‘ghost’ nuclei and very short telomeres

In Epstein–Barr virus (EBV) negative Hodgkins cell lines and classical EBV-negative Hodgkins lymphoma (HL), Reed–Sternberg cells (RS cells) represent end-stage tumor cells, in which further nuclear division becomes impossible because of sustained telomere loss, shortening and aggregation. However, the three-dimensional (3D) telomere organization in latent membrane protein 1 (LMP1)-expressing RS cells of EBV-associated HL is not known. We performed a 3D telomere analysis after quantitative fluorescent in situ hybridization on 5 μm tissue sections on two LMP1-expressing HL cases and showed highly significant telomere shortening (P<0.0001) and formation of telomere aggregates in RS cells (P<0.0001), when compared with the mononuclear precursor Hodgkin cells (H cells). Telomere-poor or telomere-free ‘ghost’ nuclei were a regular finding in these RS cells. These nuclei and their telomere content strongly contrasted with the corona of surrounding lymphocytes showing numerous midsized telomere hybridization signals. Both H cells and RS cells of two EBV-negative HL cases analyzed in parallel showed 3D telomere patterns identical to those of LMP1-expressing cases. As a major advance, our 3D nuclear imaging approach allows the visualization of hitherto unknown profound changes in the 3D nuclear telomere organization associated with the transition from LMP1-positive H cells to LMP1-positive RS cells. We conclude that RS cells irrespective of LMP1 expression are end-stage tumor cells in which the extent of their inability to divide further is proportional to the increase of very short telomeres, telomere loss, aggregate formation and the generation of ‘ghost’ nuclei.

Synthetic lethal targeting of superoxide dismutase 1 selectively kills RAD54B-deficient colorectal cancer cells.

Synthetic lethality is a rational approach to identify candidate drug targets for selective killing of cancer cells harboring somatic mutations that cause chromosome instability (CIN). To identify a set of the most highly connected synthetic lethal partner genes in yeast for subsequent testing in mammalian cells, we used the entire set of 692 yeast CIN genes to query the genome-wide synthetic lethal datasets. Hierarchical clustering revealed a highly connected set of synthetic lethal partners of yeast genes whose human orthologs are somatically mutated in colorectal cancer. Testing of a small matrix of synthetic lethal gene pairs in mammalian cells suggested that members of a pathway that remove reactive oxygen species that cause DNA damage would be excellent candidates for further testing. We show that the synthetic lethal interaction between budding yeast rad54 and sod1 is conserved within a human colorectal cancer context. Specifically, we demonstrate RAD54B-deficient cells are selectively killed relative to controls via siRNA-based silencing and chemical inhibition and further demonstrate that this interaction is conserved in an unrelated cell type. We further show that the DNA double strand breaks, resulting from increased reactive oxygen species following SOD1 inhibition, persist within the RAD54B-deficient cells and result in apoptosis. Collectively, these data identify SOD1 as a novel candidate cancer drug target and suggest that SOD1 inhibition may have broad-spectrum applicability in a variety of tumor types exhibiting RAD54B deficiencies.

Characterizing the Prevalence of Chromosome Instability in Interval Colorectal Cancer

A substantial proportion of colorectal cancers (CRCs) are interval CRCs (I-CRCs; i.e., CRCs diagnosed soon after a colonoscopy). Chromosomal instability (CIN) is defined as an increase in the rate of which whole chromosomes/large chromosomal fragments are gained or lost and is observed in 85% of non-hereditary CRCs. The contribution of CIN to the etiology of I-CRCs remains unknown. We established a fluorescence in situ hybridization (FISH) approach to characterize CIN by enumerating specific chromosomes and determined the prevalence of numerical CIN in a population-based cohort of I-CRCs and control (sporadic) CRCs. Using the population-based Manitoba Health administrative databases and Manitoba Cancer Registry, we identified an age, sex, and colonic site of CRC matched cohort of I-CRCs and controls and retrieved their archived paraffin-embedded tumor samples. FISH chromosome enumeration probes specifically recognizing the pericentric regions of chromosomes 8, 11, and 17 were first used on cell lines and then CRC tissue microarrays to detect aneusomy, which was then used to calculate a CIN score (CS). The 15th percentile CS for control CRC was used to define CIN phenotype. Mean CSs were similar in the control CRCs and I-CRCs; 82% of I-CRCs exhibited a CIN phenotype, which was similar to that in the control CRCs. This study suggests that CIN is the most prevalent contributor to genomic instability in I-CRCs. Further studies should evaluate CIN and microsatellite instability (MSI) in the same cohort of I-CRCs to corroborate our findings and to further assess concomitant contribution of CIN and MSI to I-CRCs.

The temporal dynamics of chromosome instability in ovarian cancer cell lines and primary patient samples

Epithelial ovarian cancer (EOC) is the most prevalent form of ovarian cancer and has the highest mortality rate. Novel insight into EOC is required to minimize the morbidity and mortality rates caused by recurrent, drug resistant disease. Although numerous studies have evaluated genome instability in EOC, none have addressed the putative role chromosome instability (CIN) has in disease progression and drug resistance. CIN is defined as an increase in the rate at which whole chromosomes or large parts thereof are gained or lost, and can only be evaluated using approaches capable of characterizing genetic or chromosomal heterogeneity within populations of cells. Although CIN is associated with numerous cancer types, its prevalence and dynamics in EOC is unknown. In this study, we assessed CIN within serial samples collected from the ascites of five EOC patients, and in two well-established ovarian cancer cell models of drug resistance (PEO1/4 and A2780s/cp). We quantified and compared CIN (as measured by nuclear areas and CIN Score (CS) values) within and between serial samples to glean insight into the association and dynamics of CIN within EOC, with a particular focus on resistant and recurrent disease. Using quantitative, single cell analyses we determined that CIN is associated with every sample evaluated and further show that many EOC samples exhibit a large degree of nuclear size and CS value heterogeneity. We also show that CIN is dynamic and generally increases within resistant disease. Finally, we show that both drug resistance models (PEO1/4 and A2780s/cp) exhibit heterogeneity, albeit to a much lesser extent. Surprisingly, the two cell line models exhibit remarkably similar levels of CIN, as the nuclear areas and CS values are largely overlapping between the corresponding paired lines. Accordingly, these data suggest CIN may represent a novel biomarker capable of monitoring changes in EOC progression associated with drug resistance.

Sister chromatid cohesion defects are associated with chromosome instability in Hodgkin lymphoma cells

BackgroundChromosome instability manifests as an abnormal chromosome complement and is a pathogenic event in cancer. Although a correlation between abnormal chromosome numbers and cancer exist, the underlying mechanisms that cause chromosome instability are poorly understood. Recent data suggests that aberrant sister chromatid cohesion causes chromosome instability and thus contributes to the development of cancer. Cohesion normally functions by tethering nascently synthesized chromatids together to prevent premature segregation and thus chromosome instability. Although the prevalence of aberrant cohesion has been reported for some solid tumors, its prevalence within liquid tumors is unknown. Consequently, the current study was undertaken to evaluate aberrant cohesion within Hodgkin lymphoma, a lymphoid malignancy that frequently exhibits chromosome instability.MethodsUsing established cytogenetic techniques, the prevalence of chromosome instability and aberrant cohesion was examined within mitotic spreads generated from five commonly employed Hodgkin lymphoma cell lines (L-1236, KM-H2, L-428, L-540 and HDLM-2) and a lymphocyte control. Indirect immunofluorescence and Western blot analyses were performed to evaluate the localization and expression of six critical proteins involved in the regulation of sister chromatid cohesion.ResultsWe first confirmed that all five Hodgkin lymphoma cell lines exhibited chromosome instability relative to the lymphocyte control. We then determined that each Hodgkin lymphoma cell line exhibited cohesion defects that were subsequently classified into mild, moderate or severe categories. Surprisingly, ~50% of the mitotic spreads generated from L-540 and HDLM-2 harbored cohesion defects. To gain mechanistic insight into the underlying cause of the aberrant cohesion we examined the localization and expression of six critical proteins involved in cohesion. Although all proteins produced the expected nuclear localization pattern, striking differences in RAD21 expression was observed: RAD21 expression was lowest in L-540 and highest within HDLM-2.ConclusionWe conclude that aberrant cohesion is a common feature of all five Hodgkin lymphoma cell lines evaluated. We further conclude that aberrant RAD21 expression is a strong candidate to underlie aberrant cohesion, chromosome instability and contribute to the development of the disease. Our findings support a growing body of evidence suggesting that cohesion defects and aberrant RAD21 expression are pathogenic events that contribute to tumor development.

KIF11 silencing and inhibition induces chromosome instability that may contribute to cancer

Understanding the aberrant pathways that contribute to oncogenesis and identifying the altered genes involved in these pathways is a critical first step to develop effective strategies to better combat cancer. Chromosome instability (CIN) is an aberrant phenotype that occurs in ∼80% of all cancer types and is associated with aggressive tumors, the acquisition of multidrug resistance and poor patient prognosis. Despite these associations however, the aberrant genes and molecular defects underlying CIN remain poorly understood. KIF11 is an evolutionarily conserved microtubule motor protein that functions in centrosome and chromosome dynamics in mitosis. Interestingly, the yeast ortholog of KIF11, namely CIN8 is a CIN gene and thus aberrant KIF11 expression and function is suspected to underlie CIN. In support of this possibility, KIF11 is somatically altered in a large number of cancer types. Using a complementary biochemical and genetic approach we examined whether KIF11 silencing with siRNAs or inhibition with monastrol was able to convert two distinct and karyotypically stable cell lines into karyotypically unstable cell lines. Indeed, quantitative imaging microscopy and flow cytometry revealed that KIF11 silencing induced increases in nuclear areas, micronucleus formation, DNA content and chromosome numbers relative to controls that was also observed following KIF11 inhibition. Collectively, this study identifies and validates KIF11 as an evolutionarily conserved CIN gene, and further suggests that aberrant expression and function may contribute to the pathogenesis of a subset of cancers.

Abstract 3587: Multiplexed nuclear area and micronucleus screening identifies SKP1 as a human chromosome instability gene

Chromosome instability (CIN) is defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost. It is hallmark of cancer that occurs frequently in both solid and liquid tumors. In addition, CIN is associated with highly aggressive tumors, the acquisition of multi-drug resistance, tumor recurrence and poor patient prognosis. Despite this, the majority of human CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. In this study we utilized two complementary, image-based approaches capable of detecting CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. The first assay involves quantifying nuclear areas following silencing, where changes in mean nuclear area relative to controls act as a surrogate marker of CIN. The second approach monitors micronucleus (MN) formation where increases in the number of micronuclei are indicative of DNA damage or the mitotic defects that underlie CIN. These assays were employed in a high-content screen of 164 human candidate CIN genes in two unrelated cell lines, HT1080 and hTERT. In HT1080, the nuclear area and MN enumeration assays identified 88 and 96 putative CIN genes, respectively. In hTERT, the nuclear area and MN assays identified 112 and 19 putative CIN genes, respectively. Promising putative CIN genes such as SKP1 were identified and prioritized for subsequent validation based on the number of assays that identified the gene, and the strength of the CIN phenotype. Preliminary data collected through Western blotting, mitotic chromosome spreads and flow cytometry, provides evidence to support the validation of SKP1 as a bona fide human CIN gene. Identification and characterization of human CIN genes will provide critical insights into CIN and tumorigenesis, as well as identify potential targets that could be exploited in novel, precision medicine approaches for superior cancer treatment. Citation Format: Laura Thompson, Allison Baergen, Zelda Lichtensztejn, Kirk McManus. Multiplexed nuclear area and micronucleus screening identifies SKP1 as a human chromosome instability gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3587.

Abstract 3585: KIF11 silencing or inhibition induces chromosome instability

Chromosome Instability (CIN) is defined as an increase in the rate at which whole chromosomes or large parts are gained or lost. CIN is not only associated with virtually all tumor types, but it is associated with aggressive tumors, the acquisition of multi-drug resistance and consequently poor patient prognosis. Despite these associations, the genes and molecular defects that contribute to CIN are only poorly understood. Recently, we performed a high content screen that identified KIF11, a microtubule associated motor protein, as a candidate CIN gene. Here, we couple RNAi-based gene silencing with biochemistry and cell biology to show that diminished KIF11 expression and/or function induce CIN. HCT116 cells were employed, as they are a karyotypically stable colorectal cancer cell line of epithelial origin that has been used for similar CIN studies. KIF11 was either silenced (both individual and pooled siRNA duplexes) or inhibited (Monostrol) and expression levels were determined by Western blots. To determine whether KIF11 silencing or inhibition affects DNA content, two phenotypes frequently associated with CIN, namely increases in nuclear area and micronucleus formation were evaluated. Fluorescence microscopy was employed on DAPI-counterstained samples and revealed statistically significant increases both nuclear area and micronucleus formation following KIF11 silencing and inhibition relative to controls. Next, flow cytometry was performed on propidium iodide labeled samples to assess whether increases in DNA content were associated with the changes in nuclear area. As predicted, increases in the proportion of cells with >G2/M DNA content occurred within the KIF11 silenced populations. Finally, mitotic chromosome spreads were generated and chromosomes were manually enumerated from 100 spreads per condition/control. Subsequent Kolmogorov-Smirnov tests identified statistically significant increases in the cumulative distribution frequencies of mitotic chromosome numbers within the spreads generated from the KIF11 silenced cells relative to controls. To extend our findings beyond the colorectal cancer cell context employed above, analogous studies were performed in hTERT cells (karyotypically stable fibroblast cell line) with very similar results. Collectively, these data indicate that KIF11 expression and function are normally required to maintain genome integrity. They further suggest that the loss of KIF11 expression and/or function may be a contributing factor in the etiology of tumorigenesis in colorectal cancer and perhaps other tumor types as well. Citation Format: Yasamin Asbaghi, Zelda Lichtensztejn, Laura Thompson, Kirk McManus. KIF11 silencing or inhibition induces chromosome instability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3585.

Abstract 3034: A high-content screen to identify novel chromosome instability genes

Chromosome instability (CIN) is defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost. It is a characteristic of virtually all cancer types that is frequently observed in highly aggressive, drug resistant tumors. Despite this, the majority of human CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. In this study we developed image-based approaches capable of detecting CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. The first assay involves quantifying nuclear areas following silencing, where changes in mean nuclear area relative to controls act as a surrogate marker of CIN. The second approach monitors micronucleus (MN) formation where increases in the number of micronuclei are indicative of CIN. These assays were employed in a high-content screen of 164 human candidate CIN genes in two unrelated cell lines, HT1080 and hTERT. In HT1080, the nuclear area and MN enumeration assays identified 43 and 83 putative CIN genes, respectively. In hTERT, the nuclear area and MN assays identified 55 and 48 putative CIN genes, respectively. Preliminary data collected through Western blotting, mitotic spreads and flow cytometry, has provided evidence to support the validation of a subset of these putative CIN genes (e.g. SKP1), as bona fide human CIN genes. Identifying novel CIN genes will provide critical insights into CIN and tumorigenesis, as well as identify potential targets that could be exploited for the development of superior therapeutic strategies. Citation Format: Laura L. Thompson, Allison Baergen, Zelda Lichtensztejn, Kirk J. McManus. A high-content screen to identify novel chromosome instability genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2015-3034