Fragile Site Gene Research Articles

Abstract 222: High frequency of structural variants in FFPE colorectal cancer tissue detected by targeting selected common fragile site genes and LINE transposable elements

Abstract Introduction: Structural variants (SVs) caused by chromosomal rearrangements or LINE retrotranspositions are a highly prevalent type of somatic DNA alterations in colorectal cancer (CRC). Studies about the function of SVs in tumor biology and their putative application as biomarkers are currently hampered by the need for fresh frozen tumor material to detect SVs by long-read or deep whole genome sequencing. This impedes the identification of SVs in retrospective cohorts and implementation in routine diagnostics (where only formalin-fixed paraffin-embedded (FFPE) tissue is available). In this study, we explored the applicability of FFPE-Targeted Locus Capture (FFPE-TLC) as a novel technology for targeted detection of SVs in CRC. Materials and methods: We defined the regions most frequently presenting SVs in CRC and included these in a customized capture panel. FFPE-TLC followed by targeted sequencing was performed on primary tumors of 29 metastatic CRC patients and eight patient-matched normal colon tissues. SV-specific droplet digital PCR (ddPCR) assays were designed for orthogonal validation of SVs in tumor tissue and detection of SVs in cell-free plasma circulating tumor DNA (ctDNA). Results: The regions associated with most SVs in metastatic CRC were four common fragile site (CFS) genes; MACROD2, PARK2, FHIT, WWOX, and three LINE source elements, and were included in a 2.7 Mb targeted panel. After filtering for germline variation, we identified 168 somatic SVs in 26/29 patients (90%), median SVs per patient: 3, range: 1-22. 110 SVs were found in CFS genes in 19/29 patients (66%), 12 of them presenting SVs in MACROD2. 58 SVs were caused by LINE integrations in 19/29 patients (66%), with different LINE behaviors observed: one source LINE generated 19 SVs in 13 patients, whilst another LINE caused 33 SVs in 5 patients. For selected cases, the tumor-specific origin of SVs was verified and its applicability as a plasma ctDNA biomarker demonstrated. Discussion: This study demonstrates, for the first time, the feasibility of targeted detection of SVs in CFS genes and SVs caused by LINE retrotransposition in routinely obtained FFPE tissue without prior knowledge of the breakpoint location. The high prevalence of SVs offers opportunities to detect many somatic alterations by targeting few genes and LINEs, e.g. for translation into ctDNA biomarker assays. We conclude that FFPE-TLC enables the investigation of SV mutations in retrospective cohorts and opens new avenues for mutation analysis in routine diagnostics. Citation Format: Carmen Rubio-Alarcon, Ellen Stelloo, Daan C. Vessies, Iris van ‘t Erve, Nienke Mekkes, Joost Swennenhuis, Soufyan Lakbir, Elise van Bree, Marianne Tijssen, Pien Delis-van Diemen, Mirthe Lanfermeijer, Theodora C. Linders, Daan van den Broek, Cornelis J. Punt, Jaap Heringa, Gerrit A. Meijer, Sanne Abeln, Harma Feitsma, Remond J. Fijneman. High frequency of structural variants in FFPE colorectal cancer tissue detected by targeting selected common fragile site genes and LINE transposable elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 222.

WWOX activates autophagy to alleviate lipopolysaccharide-induced acute lung injury by regulating mTOR.

Acute lung injury (ALI) is characterized by acute systemic inflammatory responses that may lead to severe acute respiratory distress syndrome (ARDS). The clinical course of ALI/ARDS is variable; however, it has been reported that lipopolysaccharides (LPS) play a role in its development. The fragile chromosomal site gene WWOX is highly sensitive to genotoxic stress induced by environmental exposure and is an important candidate gene for exposure-related lung disease research. However, the expression of WWOX and its role in LPS-induced ALI still remain unidentified. This study investigated the expression of WWOX in mouse lung and epithelial cells and explored the role of WWOX in LPS-induced ALI model in vitro and in vivo. In addition, we explored one of the possible mechanisms by which WWOX alleviates ALI from the perspective of autophagy. Here, we observed that LPS stimulation reduced the expression of WWOX and the autophagy marker microtubule-associated protein 1 light chain 3β-II (MAP1LC3B/LC3B) in mouse lung epithelial and human epithelial (H292) cells. Overexpression of WWOX led to the activation of autophagy and inhibited inflammatory responses in LPS-induced ALI cells and mouse model. More importantly, we found that WWOX interacts with mechanistic target of rapamycin [serine/threonine kinase] (mTOR) and regulates mTOR and ULK-1 signaling-mediated autophagy. Thus, reduced WWOX levels were associated with LPS-induced ALI. WWOX can activate autophagy in lung epithelial cells and protect against LPS-induced ALI, which is partly related to the mTOR-ULK1 signaling pathway.

Abstract A020: Structural variants in the pathogenesis of colorectal cancer: The elephant in the room

Abstract Background: Cancer is caused by somatic DNA alterations, comprising single/small nucleotide variants (SNVs), somatic copy number alterations (SCNAs) and chromosomal rearrangement structural variants (SVs). We previously demonstrated that SVs are recurrently identified in hundreds of genes and are highly prevalent in common fragile site genes, e.g., in MACROD2 in >40% of colorectal cancers (CRCs). However, computational methods that discriminate SV-driver from SV-passenger events are lacking and laboratory methods to detect SVs at nucleotide resolution from routinely obtained formalin-fixed paraffin-embedded (FFPE) tumor tissue material are underdeveloped. Therefore, despite the abundant presence of SVs, knowledge about their biological and clinical impact is limited. Aim: The aim of our studies is to identify genes of which the function is frequently affected by SV, to understand how these genes contribute to CRC pathogenesis, and to translate these SVs into clinically relevant biomarkers. Methods: We made use of publicly available deep whole genome DNA sequencing data and tumor-matched RNA sequencing data from the Hartwig Medical Foundation to develop the algorithm ‘CoBRA’: Computation of Biologically Relevant Alterations. Adenoma-derived organoids were used for CRISPR/Cas9-mediated gene modulation for functional analysis of SV-driver events. Cergentis’ targeted locus capture (FFPE-TLC) technology was used to detect SVs at nucleotide resolution from FFPE material, which were translated into droplet digital PCR (ddPCR) assays for the detection of SVs in cell-free circulating tumor DNA (ctDNA) in liquid biopsies. Results: The CoBRA algorithm associated the presence of SV-events in frequently affected genes to the extent in which genome-wide RNA sequencing data were altered. In this way, CoBRA ranked SV-events in genes according to their putative impact on tumor biology. SVs in MACROD2 ranked among those with the highest impact on tumor biology. Therefore, we generated focal deletions in MACROD2 in adenoma-derived organoids for functional analyses. Moreover, using FFPE tumor tissue material we detected SVs at nucleotide resolution in MACROD2 and three other genes in 21 out of 29 patients. SVs were verified by PCR on tumor tissue and subsequently translated into ddPCR biomarker assays for detection of SVs in ctDNA in blood from the same patients. Conclusions: We developed the computational method CoBRA and succeeded to detect SVs with high impact on tumor biology. These SVs are prioritized for functional analysis in pre-malignant adenoma-derived organoids; for targeted detection in routinely obtained FFPE tumor tissue material; and for translation into liquid biopsy ctDNA assays. Proof of concept was delivered for MACROD2. Our novel computational and laboratory methodologies provide valuable tools to effectively explore the biological and clinical impact of SVs, which will contribute to our understanding of these common recurrent somatic alterations in CRC and their translation into clinically relevant biomarker applications. Citation Format: Elise van Bree, Carmen Rubio Alarcón, Soufyan Lakbir, Ellen Stelloo, Caterina Buranelli, Amber Hondema, Iris van 't Erve, Daan Vessies, Pien Delis-van Diemen, Marianne Tijssen, Anne Bolijn, Mirthe Lanfermeijer, Dorothe Linders, Joost Swennenhuis, Daan van den Broek, Jaap Heringa, Gerrit Meijer, Beatriz Carvalho, Harma Feitsma, Sanne Abeln, Remond J. A. Fijneman. Structural variants in the pathogenesis of colorectal cancer: The elephant in the room [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A020.

Large Intronic Deletion of the Fragile Site Gene PRKN Dramatically Lowers Its Fragility Without Impacting Gene Expression

Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.

Cigarette Smoke and Nicotine-Containing Electronic-Cigarette Vapor Downregulate Lung WWOX Expression, Which Is Associated with Increased Severity of Murine Acute Respiratory Distress Syndrome.

A history of chronic cigarette smoking is known to increase risk for acute respiratory distress syndrome (ARDS), but the corresponding risks associated with chronic e-cigarette use are largely unknown. The chromosomal fragile site gene, WWOX, is highly susceptible to genotoxic stress from environmental exposures and thus an interesting candidate gene for the study of exposure-related lung disease. Lungs harvested from current versus former/never-smokers exhibited a 47% decrease in WWOX mRNA levels. Exposure to nicotine-containing e-cigarette vapor resulted in an average 57% decrease in WWOX mRNA levels relative to vehicle-treated controls. In separate studies, endothelial (EC)-specific WWOX knockout (KO) versus WWOX flox control mice were examined under ARDS-producing conditions. EC WWOX KO mice exhibited significantly greater levels of vascular leak and histologic lung injury. ECs were isolated from digested lungs of untreated EC WWOX KO mice using sorting by flow cytometry for CD31+ CD45-cells. These were grown in culture, confirmed to be WWOX deficient by RT-PCR and Western blotting, and analyzed by electric cell impedance sensing as well as an FITC dextran transwell assay for their barrier properties during methicillin-resistant Staphylococcus aureus or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to cells from WWOX flox controls during either methicillin-resistant S. aureus or LPS treatment as measured by both electric cell impedance sensing and the transwell assay. The increased risk for ARDS observed in chronic smokers may be mechanistically linked, at least in part, to lung WWOX downregulation, and this phenomenon may also manifest in the near future in chronic users of e-cigarettes.

Complex structural rearrangements are present in high-grade dysplastic Barrett\u2019s oesophagus samples

BackgroundOesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett’s oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals.MethodsIn this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples.ResultsWe observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples.ConclusionsThe presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.

WWOX, large common fragile site genes, and cancer.

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.

Prognostic Significance of Decreased Expression of Six Large Common Fragile Site Genes in Oropharyngeal Squamous Cell Carcinomas

Common fragile sites (CFSs) are large regions with profound genomic instability that often span extremely large genes a number of which have been found to be important tumor suppressors. RNA sequencing previously revealed that there was a group of six large CFS genes which frequently had decreased expression in oropharyngeal squamous cell carcinomas (OPSCCs) and real-time reverse transcriptase polymerase chain reaction experiments validated that these six large CFS genes (PARK2, DLG2, NBEA, CTNNA3, DMD, and FHIT) had decreased expression in most of the tumor samples. In this study, we investigated whether the decreased expression of these genes has any clinical significance in OPSCCs. We analyzed the six CFS large genes in 45 OPSCC patients and found that 27 (60%) of the OPSCC tumors had decreased expression of these six genes. When we correlated the expression of these six genes to each patient’s clinical records, for 11 patients who had tumor recurrence, 10 of them had decreased expression of almost all 6 genes. When we divided the patients into two groups, one group with decreased expression of the six genes and the other group with either slight changes or increased expression of the six genes, we found that there is significant difference in the incidence of tumor recurrence between these two groups by Kaplan-Meier plot analysis (P < .05). Our results demonstrated that those OPSCC tumors with decreased expression of this select group of six large CFS genes were much more likely to be associated with tumor recurrence and these genes are potential prognostic markers for predicting tumor recurrence in OPSCC.

Very large common fragile site genes and their potential role in cancer development.

Common fragile sites (CFSs) are large chromosomal regions that are hot-spots for alterations especially within cancer cells. The three most frequently expressed CFS regions (FRA3B, FRA16D and FRA6E) contain genes that span extremely large genomic regions (FHIT, WWOX and PARK2, respectively), and these genes were found to function as important tumor suppressors. Many other CFS regions contain extremely large genes that are also targets of alterations in multiple cancers, but none have yet been demonstrated to function as tumor suppressors. The loss of expression of just FHIT or WWOX has been found to be associated with a worse overall clinical outcome. Studies in different cancers have revealed that some cancers have decreased expression of multiple large CFS genes. This loss of expression could have a profound phenotypic effect on these cells. In this review, we will summarize the known large common fragile site genes and discuss their potential relationship to cancer development.

Abstract 582: Human papillomavirus and the inactivation of expression of multiple large common fragile site genes in oropharyngeal squamous cell carcinoma

Abstract Human papillomavirus (HPV) appears to be responsible for dramatic increases in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) in the United States in the last two decades, in spite of decreases in the incidence of smoking. It is generally assumed that the role of HPV in the development of OPSCC is identical to the one that it plays in cervical cancer where HPV infection followed by integration into the host genome and the subsequent increased expression of the E6 and E7 oncoproteins leads to increased genomic instability. The common fragile sites (CFSs) are highly unstable chromosomal regions that will be particularly sensitive to increased genomic instability and they are also hot-spots for viral integrations in cervical cancer. The CFS regions span many megabases and many of them also span extremely large genes, a number of which have been demonstrated to function as important tumor suppressors. We therefore examined the expression of very large genes in OPSCC. An analysis of RNAseq data generated on 11 OPSCCs and matched normal oropharyngeal tissue from the same patients revealed that some of the tumors had greatly decreased expression for many of the largest genes. The two genes with the greatest decreases in expression in the tumors as compared to matched normal were the known tumor suppressor PARK2 and the putative tumor suppressor DLG2, but three other large CFS genes: CTNNA3, LRP1B and DMD, and one large gene not yet precisely localized within a CFS region: PDE4D, also had much more decreased expression than any of the other large genes. Real-time RT-PCR was used to measure the expression of these six genes in a much larger number of OPSCC tumor-normal pairs. The expression of these six genes was concordant in most OPSCCs. An individual tumor generally had either decreased expression of these six genes, no changes in the expression of these genes or increased expression of all six genes. When the tumors were grouped with respect to which ones were HPV-positive or HPV-negative we observed that 40% of the HPV-positive OPSCCs had greatly decreased expression (more than 100-fold) for all six genes. Half of the HPV-negative tumors had decreased expression of these six genes, but the amount of decreased expression was much more modest (4-16 fold). Most exciting, however was that 7 of the 12 HPV-positive tumors, and three of the four of the HPV-negative patients that had decreased expression of these genes were from patients who had tumor recurrence. In contrast only one of 18 patients whose tumors did not have decreased expression of the large genes had tumor recurrence. The loss of expression of multiple large genes, many of which are derived from CFS regions, could thus be a powerful predictor for which patients will have tumor recurrence. These results also suggest a linkage between HPV-induced chromosomal instability and dramatic decreases in the expression of multiple CFS genes in some OPSCCs. Citation Format: David I. Smith, Ge Gao, Nicole Tombers, Jan Kasperbauer, Vivian Wang. Human papillomavirus and the inactivation of expression of multiple large common fragile site genes in oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 582. doi:10.1158/1538-7445.AM2014-582

A selected group of large common fragile site genes have decreased expression in oropharyngeal squamous cell carcinomas

The common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. The frequent deletions and other alterations in these regions in multiple cancers has led to the discovery of a number of extremely large genes contained within these regions and several of the large CFS genes have already been demonstrated to function as tumor suppressors involved in the formation of many different cancers. To study the large CFS genes in oropharyngeal squamous cell carcinoma (OPSCC), we did RNA seq analysis from 11 head and neck cancer patients. This revealed that there are six large CFS genes which consistently had decreased expression in the tumor samples compared to their matched normal tissues. These six genes are PARK2, DLG2, NBEA, CTNNA3, DMD, and FHIT. PARK2 and FHIT are located within the two most frequently expressed CFSs and both have been demonstrated to function as tumor suppressors, while the other large genes are found to have frequent alterations in multiple cancers. Validation experiments using real time PCR indicated that over 60% of OPSCC tumors showed decreased expression for all six genes. Both HPV-positive and HPV-negative OPSCCs had similar proportions with loss of expression of these genes. Our results suggest that this selected group of large genes might serve as potential tumor suppressors involved in the development of OPSCCs. Further studies are needed to investigate whether the decreased expression observed is due to genomic instability within the CFS regions or the selection for alterations of specific large CFS genes.

Common Chromosomal Fragile Site Gene WWOX in Metabolic Disorders and Tumors.

WWOX, a gene that spans the second most common chromosomal fragile site (FRA16D), often exhibits homozygous deletions and translocation breakpoints under multiple cellular stresses induced by extrinsic or intrinsic factors, such as hypoxia, UV, and DNA damage regents. Loss of WWOX is closely related to genomic instability, tumorigenesis, cancer progression and therapy resistance. WWOX heterozygous knockout mice show an increased incidence of spontaneous or induced tumors. WWOX can interact via the WW domain with proteins that possess proline PPxY motifs and is involved in a variety of cellular processes. Accumulating evidence has shown that WWOX that contains a short-chain dehydrogenase/reductase (SDR) domain is involved in steroid metabolism and bone development. Reduced or lost expression of WWOX will lead to development of metabolic disease. In this review, we focus on the roles of WWOX in metabolic disorders and tumors.

Efficient targeting of FATS at a common fragile site in mice through TALEN-mediated double-hit genome modification

Transcription activator-like effector nucleases (TALENs) have emerged as a newly developed approach for genome editing. However, its application in targeting specific genomic loci susceptible to DNA damage remains obscure. Here, we report a modified approach for TALENs-based targeting of FATS, a fragile-site gene whose major introns have AT-rich sequence and di-nucleotide repeats. Two pairs of FATS-TALENs were designed to cleave two sites specifically at a coding exon of FATS. After in vitro transcription, the mRNA from FATS-TALEN pairs was microinjected into mouse zygotes. The targeting efficiency of two FATS-TALEN pairs in vivo was more than threefold higher than that of one FATS-TALEN pair. Moreover, large-size DNA deletions were detected, which were heritable and easily detectable by PCR. Our study indicates that the double-hit TALEN approach enhances targeting efficiency in vivo and provides convenience for monitoring germline transmission of mutations by PCR, which will facilitate the functional research on fragile-site genes.

Common chromosomal fragile site FRA16D tumor suppressor WWOX gene expression and metabolic reprograming in cells

The WWOX gene spans the FRA16D common chromosomal fragile site and is able to suppress tumor growth. FRA16D is a frequent site of DNA instability in cancer resulting in reduced levels of WWOX expression. Altered levels of WWOX have been shown to affect metabolism. Whereas metabolic reprograming of cells from oxidative phosphorylation to aerobic glycolysis is a major hallmark of tumors, the relationship between common chromosomal fragile site genes and altered metabolism has been unclear. Here we report that altering metabolism from glycolysis to oxidative phosphorylation causes stable increase in steady-state levels of transcripts of the WWOX gene. Consistent with this, exposure to hypoxic conditions, in which cells rely on glycolysis, causes a downregulation of WWOX mRNA. The function of WWOX is therefore intimately integrated with metabolism, as WWOX not only contributes to the metabolic state of cells, its transcript levels are also linked to intracellular metabolic state.

Common Fragile Site Gene FRA16D WWOX (WW domain containing oxidoreductase) Is Frequently Altered in Plasma Cells of Multiple Myeloma and MGUS, Possibly Related to Disease Development

Abstract 4646The common fragile sites (CFSs) are regions of profound genomic instability, and hot-spots for deletions and other alterations in cancer cells. We have previously shown that promoter of FHIT is frequently methylated in multiple myeloma (MM) and correlated with worse prognosis. WWOX is located at a CFS region on chromosome 16q23.3, this gene is also a target of alterations in multiple cancers. The objective of current study is to find if this tumor suppressor gene is altered in MM and MGUS. Six myeloma cell lines, bone marrow mononuclear cells (BMMNC) of 165 MM and 25 MGUS patients were subjected to this study after obtaining informed consent. Isolated CD138 positive plasma cells (PC) of 24 MM were examined to see if the alteration occurred really in myeloma cells. Using methylation specific PCR, WWOX promoter methylation was detected in 2 of 6 cell lines, 35% of MM patients and 8% of MGUS patients (p=0.01). The overall survival of the MM patients with methylated WWOX tended to be worse than with unmethylated WWOX (p=0.2). Using nested RT-PCR, aberrant short transcripts of WWOX lacking exons coding SDR domain were detected in 4 cell lines, 68% of MM and 60% of MGUS (p=0.51) In isolated PC, aberrant transcripts of WWOX were detected in 58% of MM and 25% of MGUS (p=0.02). Real time PCR demonstrated higher WWOX expression in isolated PC of MM and MGUS than of BMMNC other than plasma cells (p=0.0001), and those higher WWOX were aberrant type (p=0.001). As aberrant WWOX is known to function as dominant negative for wild type, our results imply that this aberrant WWOX is an oncogene as well as losing tumor suppressor function. Since WWOX is demonstrated to regulate beta-catenin and NF-kB pathway, high frequency of aberrant WWOX expression indicates an important role in myeloma development. Disclosures:No relevant conflicts of interest to declare.

Gephyrin and neurexin 3 are new large common fragile site (cFS) genes frequently rearranged in different human cancers

Common fragile sites (cFS) are regions of genomic instability that are prone to breakage under conditions partially inhibiting DNA synthesis. These regions are found in all individuals and appear to be hotspots of chromosomal rearrangements in cancers and neurological diseases. It has been suggested that DNA damage during the early stages of tumorigenesis preferentially targets cFSs, since they are sensitive to replication stress. The identification of the full repertoire of Human FRAGILOME sequences is an important task of Cancer Genome research leading to the discovery of new cancer susceptibility genes.

Abstract 254: A HDAC1-binding domain within FATS bridges p21 turnover to radiation-induced tumorigenesis

Abstract There is a gap between the initial formation of cells carrying radiation-induced genetic damage and their contribution to cancer development. Herein we reveal a previously uncharacterized gene FATS through a genome-wide approach and demonstrate its essential role in regulating the abundance of p21 in surveillance of genome integrity. A large exon coding the NH2-terminal domain of FATS, deleted in spontaneous mouse lymphomas, is much more frequently deleted in radiation-induced mouse lymphomas. Its human counterpart is a fragile site gene at a previously identified loss of heterozygosity (LOH) site. FATS is essential for maintaining steady-state level of p21 protein and sustaining DNA damage checkpoint. Furthermore, the NH2-terminal FATS physically interacts with HDAC1 to enhance the acetylation of endogenous p21, leading to the stabilization of p21. Our results reveal a molecular linkage between p21 abundance and radiation-induced carcinogenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 254.

Abstract 251: Disabled gene is involved in CML progression and its expression level at diagnosis can predict major molecular response (MMR) to imatinib therapy

Abstract Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. Using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we identified Dab1 and Dab2, the homologs of Dm Disabled (Dab), as genes involved in CML progression. In Dm the Dab loss of function induced a worsening of the hBcr-Abl eye phenotype and an even stronger phenotype was obtained using Dab RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Dab is an adaptor protein acting downstream of many receptor tyrosine kinases (RTK). One of the human homolog of Dab, Dab1 is a large common fragile site gene involved in neural migration, and the other homolog Dab2 encodes an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many cancers suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM) distributed as follows: 55 patients at diagnosis (19 enrolled in TOPS study), 9 chronic phase (CP), 7 accelerated phase (AP) and 11 blast crisis (BC). 21 healthy donors (10 PB and 11 BM) were analyzed as control. In 18 patients, genes expression was analyzed during remission as well. Protein expression was evaluated by Western Blot (WB) and Immunofluorescence (IF). In addition, K562 cells were transfected with Dab plasmids to evaluate the effects on cell proliferation. We found that in CML patients Dab1/2 expression was significantly decreased both in BM and PB (p&amp;lt;0.002 and p&amp;lt;0.0004) compared to healthy donors. In BC Dab1/2 levels were further decreased whereas during remission the expression was comparable to normal values. Data analysis of patients included in TOPS studies shows that Dab1 values are higher among those achieving MMR by 12 months (median value: 0,017) compared to those without MMR (median value: 0,001); WB and IF confirmed the absence of Dab1/2 proteins in course of active CML samples while it reappeared during remission. Moreover, Dab1 transfection of K562 significantly reduced proliferation (p=0,002). In conclusion, our results show a significant decrease of Dab1/2 expression in BC samples, when compared to CP CML and healthy donors. Among CP CML patients the responders to Tyrosine Kinase Inhibitors (TKI) therapy have been detected to express higher Dab levels than non responders, and these expression levels can predict MMR to Imatinib therapy. In conclusion, this study points to specific gene pathways that might offer new molecular markers for the monitoring of CML and new targets for CML therapy in order to prevent or overcome disease progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 251.

A novel role of IκBα and WWOX in promoting lymphoid Molt-4 and Jurkat T cell differentiation (36.42)

Abstract Human common fragile site gene WWOX, encoding WW domain-containing oxidoreductase (designated WWOX, FOR or WOX1), plays critical roles in the pathogenesis of cancer and maintenance of normal physiology. To explore the role of WOX1 in lymphoid cancer, here we determined that transient expression of WOX1 induced the expression of a differentiation marker CD3 in an acute lymphoblastic leukemia cell line Molt-4, but the induction was less effective in better-differentiated Jurkat T cells. Upregulation of RORγ, one of the Th17 cell markers, was shown in Molt-4, but not Jurkat T cells, overexpressing WOX1. Mapping analysis revealed that the N-terminal WW domain of WOX1 induced T lymphoid cell differentiation, in which a dominant negative and phosphorylation mutants (i.e. Y33R and Y61R) of WOX1 blocked the effect. We determined that the N-terminal WW domain of WOX1 physically interacts with IκBα (an inhibitor of NF-κB), as determined by co-immunoprecipitaiton and Fluorescence/Förster resonance energy transfer (FRET). Ectopic IκBα, via its N-terminal ankyrin-repeat region, induced Molt-4 differentiation. Both WOX1 and IκBα did not enhance T cell differentiation synergistically, suggesting that they may nullify each other’s function. In positive controls, phorbol and ionophore A23187 induced cell differentiation by upregulating IκBα, CD3 and WOX1 in Molt-4 T-cells. Together, our finding demonstrates a novel WOX1/IκBα regulatory mechanism for T-cell differentiation.

An HDAC1-binding domain within FATS bridges p21 turnover to radiation-induced tumorigenesis

There is a gap between the initial formation of cells carrying radiation-induced genetic damage and their contribution to cancer development. Herein, we reveal a previously uncharacterized gene FATS through a genome-wide approach and demonstrate its essential role in regulating the abundance of p21 in surveillance of genome integrity. A large exon coding the NH2-terminal domain of FATS, deleted in spontaneous mouse lymphomas, is much more frequently deleted in radiation-induced mouse lymphomas. Its human counterpart is a fragile site gene at a previously identified loss of heterozygosity site. FATS is essential for maintaining steady-state level of p21 protein and sustaining DNA damage checkpoint. Furthermore, the NH2-terminal FATS physically interacts with histone deacetylase 1 (HDAC1) to enhance the acetylation of endogenous p21, leading to the stabilization of p21. Our results reveal a molecular linkage between p21 abundance and radiation-induced carcinogenesis.