Monoallelic Deletion Research Articles

Abstract 4464: Biological functions of LSAMP, a gene frequently deleted in African American prostate cancers

Abstract Introduction: Most genomic studies of prostate cancer (CaP) have been performed primarily on men of European ancestry, including Caucasian Americans. African Americans (AA) men, however, bear the burden of highest CaP incidence and mortality rates in the US. Cumulative data from other groups and from us have shown significantly lower rate of alterations of the two common CaP driver genes (PTEN and ERG) in AA CaP as compared to CA CaP. Using a combination of whole genome sequencing, IHC and FISH based validations on tissue microarrays, and analysis of the TCGA data from a total of 438 CaPs, we recently reported a higher frequency of a genomic deletion on 3q13.31 locus in AA CaP which also associated with rapid biochemical recurrence. This deletion is centered at the LSAMP (Limbic System Associated Membrane Protein) gene. The LSAMP protein functions as a cell adhesion molecule affecting cell differentiation and migration during development. We hypothesize that the loss of LSAMP contributes to CaP progression. Here, we report the biological consequence of LSAMP modulation in CaP cells. Methods: LSAMP status in CaP cell lines (LNCaP, MDAPCa2b, CWR22rv1, LAPC4, NCI-H660, and PC3) was assessed by FISH, immunoblot, qRT-PCR and immuno-fluorescence (IF), assays using MC-IXC neuroepithelioma cell line as the positive control. We established doxycycline inducible LSAMP in LNCaP and in MDAPCa2b (an AA patient derived cell line with monoallelic LSAMP deletion) cells, and constitutive expression in DU145 cells. The effect of LSAMP loss and over-expression on cell growth, proliferation apoptosis and cell migration was monitored by MTT, soft-agar, and Boyden chamber assays. Results: Of the CaP cell lines analyzed, LSAMP RNA and protein expression was detected in PC3 and NCI-H660. IF assay showed that endogenously expressed LSAMP is predominanty localized to the cell surface. Exogenous expression of LSAMP, either constitutively in DU145, or by inducible means in LNCaP and MDAPCa2b cells, showed similar localization to the plasma membrane. Both inducible and constitutive overexpression of LSAMP resulted in reduced cell growth. LSAMP expression in DU145 resulted in enhanced formation of filamentous extracellular extensions. Analysis of published TCGA (The Cancer Genome Atlas) data showed a correlation of high LSAMP promoter methylation and low LSAMP expression in CaP tumors compared to normal prostate tissues. Conclusion: The cell biology features of LSAMP reported here, together with observations of genomic deletion and methylation of LSAMP promoter in CaP, support its role in CaP development or progression. These data support the function of LSAMP as a tumor suppressor gene, as previously reported in other cancers. Citation Format: Shyh-Han Tan, Kevin Babcock, Indu Kohaar, Ahmed A. Mohamed, Denise Young, Shiv Srivastava, Albert Dobi. Biological functions of LSAMP, a gene frequently deleted in African American prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4464. doi:10.1158/1538-7445.AM2017-4464

Autogenous Control of 5′TOP mRNA Stability by 40S Ribosomes

Ribosomal protein (RP) expression in higher eukaryotes is regulated translationally through the 5′TOP sequence. This mechanism evolved to more rapidly produce RPs on demand in different tissues. Here we show that 40S ribosomes, in a complex with the mRNA binding protein LARP1, selectively stabilize 5′TOP mRNAs, with disruption of this complex leading to induction of the impaired ribosome biogenesis checkpoint (IRBC) and p53 stabilization. The importance of this mechanism is underscored in 5q− syndrome, a macrocytic anemia caused by a large monoallelic deletion, which we found to also encompass the LARP1 gene. Critically, depletion of LARP1 alone in human adult CD34+ bone marrow precursor cells leads to a reduction in 5′TOP mRNAs and the induction of p53. These studies identify a 40S ribosome function independent of those in translation that, with LARP1, mediates the autogenous control of 5′TOP mRNA stability, whose disruption is implicated in the pathophysiology of 5q− syndrome.

Monoallelic Large Intragenic KRT5 Deletions Account for Genetically Unsolved Cases of Epidermolysis Bullosa Simplex

Monoallelic Large Intragenic KRT5 Deletions Account for Genetically Unsolved Cases of Epidermolysis Bullosa Simplex

Association of CDKN2A/2B deletion with homozygous deletion of TCR- γ gene in T-ALL.

e18516 Background: T-lineage acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease that arises in a multistep fashion through acquisition of several genetic aberrations. Absence of biallelic deletion of TCR-γ (ABD), a marker of immaturity, and CDKN2A/2B deletion have been identified as poor predictors of outcome in T-ALL treated with contemporary protocols. The aim of this study was to determine the correlation between ABD and CDKN2A/2B in Indian T-ALL patients. Methods: A total of 31 cases of T-ALL, diagnosed on morphology, cytochemistry and immunophenotyping, were included in the study. The age of the patients ranged from 1-50 years. The patients were investigated for CDKN2A/2B deletion by multiplex ligation dependent probe amplification (MLPA) using the commercially available probe set P383 (MRC Holland). The status of the deletion of TCR- γ was determined using quantitative assay described by Gutierrez et al.1 Results: Absence of biallelic deletion of TCR- γ gene were found in 14 (45.16%) patients. Homozygous deletion was seen in 13 (41.93%) patients while monoallelic deletion was seen in 5 (16.12%) patients. CDKN2A/2B deletions were found in (48.38%) patients. On correlation of CDKN2A/2B deletion and TCR- γ deletion, it was found that CDKN2A/2B deletion was found in 2/14 (14.28%) patients with ABD and 10/13 (76.92%) patients with homozygous deletion. Conclusions: CDKN2A/2B deletion are more common in patients with homozygous deletion of TCR- γ gene.

SMARCB1 (INI-1)-deficient Sinonasal Carcinoma: A Series of 39 Cases Expanding the Morphologic and Clinicopathologic Spectrum of a Recently Described Entity.

To more fully characterize the clinical and pathologic spectrum of a recently described tumor entity of the sinonasal tract characterized by loss of nuclear expression of SMARCB1 (INI1), we analyzed 39 SMARCB1-deficient sinonasal carcinomas collected from multiple medical centers. The tumors affected 23 males and 16 females with an age range of 19 to 89 years (median, 52). All patients presented with locally advanced disease (T3, n=5; T4, n=27) involving the sinuses (mainly ethmoid) with variable involvement of the nasal cavity. Thirty patients received surgery and/or radiochemotherapy with curative intent. At last follow-up, 56% of patients died of disease 0 to 102 months after diagnosis (median, 15), 2 were alive with disease, and 1 died of an unrelated cause. Only 9 patients (30%) were alive without disease at last follow-up (range, 11 to 115 mo; median, 26). The original diagnosis of retrospectively identified cases was most often sinonasal undifferentiated carcinoma (n=14) and nonkeratinizing/basaloid squamous cell carcinoma (n=5). Histologically, most tumors displayed either a predominantly basaloid (61%) or plasmacytoid/rhabdoid morphology (36%). The plasmacytoid/rhabdoid form consisted of sheets of tumor cells with abundant, eccentrically placed eosinophilic cytoplasm, whereas similar cells were typically rare and singly distributed in the basaloid variant. Glandular differentiation was seen in a few tumors. None of the cases showed squamous differentiation or surface dysplasia. By immunohistochemistry, the tumors were positive for pancytokeratin (97%), CK5 (64%), p63 (55%), and CK7 (48%); and they were negative for NUT (0%). Epstein-Barr virus and high-risk human papillomavirus was not detected by in situ hybridization. Immunohistochemical loss of SMARCB1 (INI1) expression was confirmed for all 39 tumors. Investigation of other proteins in the SWI/SNF complex revealed co-loss of SMARCA2 in 4 cases, but none were SMARCA4 deficient or ARID1A deficient. Of 27 tumors with SMARCB1 fluorescence in situ hybridization analysis, 14 showed homozygous (biallelic) deletions and 7 showed heterozygous (monoallelic) deletions. SMARCB1-deficient sinonasal carcinoma represents an emerging poorly differentiated/undifferentiated sinonasal carcinoma that (1) cannot be better classified as another specific tumor type, (2) has consistent histopathologic findings (albeit with some variability) with varying proportions of plasmacytoid/rhabdoid cells, and (3) demonstrates an aggressive clinical course. This entity should be considered in any difficult-to-classify sinonasal carcinoma, as correct diagnosis will be mandatory for optimizing therapy and for further delineation of this likely underdiagnosed disease.

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms

AbstractBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN.

A mechanistic evaluation of the Syrian hamster embryo cell transformation assay (pH 6.7) and molecular events leading to senescence bypass in SHE cells

The implementation of the Syrian hamster embryo cell transformation assay (SHE CTA) into test batteries and its relevance in predicting carcinogenicity has been long debated. Despite prevalidation studies to ensure reproducibility and minimise the subjective nature of the assay’s endpoint, an underlying mechanistic and molecular basis supporting morphological transformation (MT) as an indicator of carcinogenesis is still missing. We found that only 20% of benzo(a)pyrene-induced MT clones immortalised suggesting that, alone, the MT phenotype is insufficient for senescence bypass. From a total of 12 B(a)P- immortalised MT lines, inactivating p53 mutations were identified in 30% of clones, and the majority of these were consistent with the potent carcinogen’s mode of action. Expression of p16 was commonly silenced or markedly reduced with extensive promoter methylation observed in 45% of MT clones, while Bmi1 was strongly upregulated in 25% of clones. In instances where secondary events to MT appeared necessary for senescence bypass, as evidenced by a transient cellular crisis, clonal growth correlated with monoallelic deletion of the CDKN2A/B locus. The findings further implicate the importance of p16 and p53 pathways in regulating senescence while providing a molecular evaluation of SHE CTA −derived variant MT clones induced by benzo(a)pyrene.

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells.

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus(129) x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Addiction to Runx1 is partially attenuated by loss of p53 in the Eµ-Myc lymphoma model.

The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the Eμ-Myc model of Burkitt's lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary Eμ-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established Eμ-Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status.

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).

BackgroundDeletion of 13q14 is the most common cytogenetic change in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is detected in about 50 % of patients by fluorescence in situ hybridization (FISH), which can reveal presence of del(13)(q14) and mono- or biallelic deletion status without information about the size of the lost region. Array-comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) can detect submicroscopic copy number changes, loss of heterozygosity (LOH) and uniparental disomy (UPD) regions. The purpose of this study was detection of the size of del(13)(q14) deletion in our group of patients, comparing the size of the monoallelic and biallelic deletions, detection of LOH and UPD regions.ResultsWe have investigated 40 CLL/SLL patients by karyotype, FISH and CGH and SNP array. Mutational status was of immunoglobulin heavy-chain variable-region (IGVH) was also examined. The size of deletion ranged from 348,12 Kb to 38.97 Mb. Detected minimal deleted region comprised genes: TRIM13, miR-3613, KCNRG, DLEU2, miR-16-1, miR-15a, DLEU1. The RB1 deletions were detected in 41 % of cases. The average size in monoallelic 13q14 deletion group was 7,2 Mb while in biallelic group was 4,8 Mb. In two cases 13q14 deletions were located in the bigger UPD regions.ConclusionsOur results indicate that bigger deletion including RB1 or presence of biallelic 13q14 deletion is not sufficient to be considered as adverse prognostic factor in CLL/SLL. CytoSure Haematological Cancer and SNP array (8x60k) can precisely detect recurrent copy number changes with known prognostic significance in CLL/SLL as well as other chromosomal imbalances. The big advantage of this array is simultaneous detection of LOH and UPD regions during the same test.

Epigenetic regulation of nuclear PLCbeta1 and Cyclin D3 during Azacitidine treatment

The Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by alterations of the hematopoietic stem cells that lead to anemia, neutropenia, bleeding problems and infections. The evidence of a clinical correlation between the presence of a monoallelic gene deletion of Phospholipase Cβ1 (PLCβ1) and the progression of MDS to Acute Myeloid Leukemia (AML) opened new perspectives of research and treatments. Patients affected by MDS with a higher risk of AML evolution have a reduction in the expression of the nuclear PLCβ1, which is also epigenetically relevant in MDS. This strengthens the importance of PLCβ1 localization. In fact, PLCβ1 is a molecular target for hypomethylating agents, such Azacitidine (AZA)(1). High-risk MDS patients that respond to the drug showed an increased expression of nuclear PLCβ1 and its downstream target Cyclin D3 (CCND3), an induction of normal myeloid differentiation, and a better prognosis. Stemming from these data, our goal was to analyze the correlation between CCND3, PLCβ1 and AZA treatment. Firstly, we treated two different cellular lines, AML HL60 and histiocytic lymphoma U937, with AZA 5μM (Ec50 for HL60 cells) for 24 hours. Then, we used Real-Time PCR and Western blot to quantify both gene and protein expression. Moreover, we showed that CCND3 promoter has one CpG island. For this reason, it is possible that AZA could directly affect both PLCβ1 and CCND3 promoters. Therefore, we studied PLCβ1 binding to CCND3 promoter by chromatin immunoprecipitation (CHIP), before and after AZA treatment. Our results evidenced that the recruitment of PLCβ1 to CCND3 promoter is specifically increased after AZA treatment, leading to suppose that PLCβ1 could have a pivotal role in MDS with either a direct or indirect effect on cell cycle, proliferation and differentiation. These complicate relations need future deepening in order to demonstrate how PLCβ1 binding actually regulates CCND3 expression and how much this expression depends on CCND3 direct promoter demethylation and PLCβ1 control.

Bone Marrow Failure and Developmental Delay Caused By Mutations in Poly(A)-Specific Ribonuclease

Background:Deadenylation is a major mechanism that regulates RNA function and fate. Several mammalian deadenylases have been identified. Poly (A)-specific ribonuclease (PARN) is one of the major mammalian deadenylases that trims single-stranded poly (A) tails of mRNAs and oligoadenylated tails of H/ACA box snoRNAs and microRNAs. RNA biogenesis has emerged as a mechanism underlying several inherited diseases including well known inherited bone marrow failure syndromes (IBMFSs) such as Diamond Blackfan anemia, dyskeratosis congenita and Shwachman-Diamond syndrome. Little is known about the biological significance of germline mutations in PARN.Methods: Genome-wide screen for copy number alterations was used to identify causal mutations in patients with hematological and neurological manifestations. Four patients were identified with deletions in the PARN gene. Genomic, biochemical, cellular and knockdown experiments in human bone marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease.Results: We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination, and severe bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and had severely reduced PARN protein and deadenylation activity. Clonogenic assays of the patient's bone marrow cells showed reduced potential to generate hematopoietic colony. Fibroblasts from the patient with biallelic mutations showed markedly slow growth. Large proportion of the cells accumulated in the G1/G0 phase of cell cycle, suggesting impaired transition from G1 to the S phase. Cells from this patient also had impaired oligoadenylation of specific H/ACA box snoRNAs and scaRNAs, including the telomerase RNA component (TERC). Importantly, PARN-deficient patient cells manifested abnormalities in two pathways that are affected in IBMFSs: short telomeres and an aberrant ribosome profile. This combination of abnormalities is seen in patients with severe variants of dyskeratosis congenita (Hoyeraal-Hreidarsson syndrome). Knocking down PARN in human CD34+ marrow cells from healthy donors revealed marked defect in clonogenic potential. Morpholino knockdown of parn in zebrafish resulted in reduced formation of red blood cells and granulocytes.Conclusions: We report for the first time four patients from three different families with developmental delay or mental illness, who carried large monoallelic PARN deletions that have not been reported in healthy controls. This indicates that large PARN deletions in humans result in a neurological phenotype. Further, we showed that biallelic PARN mutations that results in markedly reduced protein, cause severe bone marrow failure and severe global central hypomyelination, similar to what is seen in patients with severe forms of dyskeratosis congenita.The identified defects suggest a new disease mechanism, in which PARN-deficiency disrupts the polyadenylated state of H/ACA box RNA molecules that in turn influences ribosome profile and telomere length and cause hematological and neurological defects. DisclosuresNo relevant conflicts of interest to declare.

Whole Genome and Transcriptome Analysis of Waldenstrom's Cell Lines Show Preservation of Many Important Genomic Features of Primary Disease, and Identify a Non-L265P Mutation in MYD88

Background: WM cell lines represent a vital tool for discovery of mechanisms responsible for WM pathogenesis, cell signaling, development of targeted therapeutics, and evaluation of mechanisms responsible for drug resistance. To date, there has been no comprehensive genomic and transcriptomic profiling of WM cell lines using next generation sequencing.Methods: We performed whole genome (WGS) and transcriptome sequencing (WTS) on three established (BCWM.1, MWCL-1, RPCI-WM1), and one novel (BCWM.2) cell line established from continuous culture of CD19-selected bone marrow cells from a WM patient with an immunophenotype consistent with primary WM disease (CD5-, CD10-, CD19+, CD20+, sIgM+, λ+, CD11c-, CD38+, and CD138+). Samples from primary tumor and germline from the founder of BCWM.2, along with tumor samples from 57 WM patients, as well as memory (CD19+ CD27+) and non-memory (CD19+ CD27-) B-cells from 9 healthy donors were also sequenced. WGS was performed using Complete Genomics platform, and WTS using Illumina HiSeq. Reads were aligned to KnownGene HG19/GRCh37 reference using STAR. Read counts per gene were obtained using featureCounts from Rsubread, and analyzed using voom from the edgeR/limma Bioconductor packages in R. Differential isoform expression was assessed using the Cufflinks software suite.Results: WGS revealed no cytogenetic level amplifications or deletions in BCWM.1, whereas MWCL-1 harbored monoallelic deletions in 17p and 17q, and amplifications in 11q. RPCI-WM1 showed deletions in 3p, 6q, 9p, 13q, 18q, 19q and X, with amplifications in 5p, 6p, 7q, 11q, 14q, 18q, 21q, and acquired uniparental disomies on 17p, 18q, and X. BCWM.2 matched its parent tumor profile with trisomy 3 and 12, as well as deletion of the entire arm of 6q and corresponding amplification of 6p. All 4 cell lines were wild type for CXCR4 and all but BCWM.2 harbored the MYD88 L265P mutation. Like its founding clone, BCWM.2 carried an activating heterozygous MYD88 G/A S243N mutation (Nature 470(7332):115-9) previously described in ABC DLBCL. In addition, a novel I318N mutation in LYN that was predicted to be activating was found in BCWM.2, but not in the founder primary tumor or germline samples. While WTS analysis of RPCI-WM1 is pending, principal component analysis showed that BCWM.1, BCWM.2 and MWCL-1 cells clustered with primary WM tumor cells, and were distinct from memory and non-memory B-cell samples from healthy donors. Likewise, multidimensional scaling of pairwise similarity clustered BCWM.2 directly with its corresponding primary sample (Figure 1). BCWM.1, BCWM.2, and MWCL-1 cells showed a high degree of recapitulation with primary MYD88mutated CXCR4wild-type WM cells including IL17RB, CABLES1, WNT5A, GPER, or WNK2 over-expression. As a group, WM cell lines were found to over-express many cancer associated genes, including MUC13, MMP7, LMO3, IL4I1, and several SRC family kinases.Conclusions: This study provides the first ever comprehensive genomic and transcriptomic profiling of WM cell lines by next generation sequencing. The findings show preservation of many important genomic features of primary WM disease, and identified the existence of a non-L265P activating MYD88 mutation, as well as dysregulation of many cancer associated genes in WM. The findings provide an important new resource for the study of WM disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Conditional knockout of tissue factor pathway inhibitor 2 in vascular endothelial cells accelerates atherosclerotic plaque development in mice

BackgroundTissue factor pathway inhibitor-2 (TFPI-2) regulates matrix metalloproteinases activation and extracellular matrix degradation. Over-expression of TFPI-2 enhances atherosclerotic plaque stability. The aim of this study is to investigate the effect of conditional knockout (KO) of TFPI-2 in vascular endothelial cells on the initiation and development of atherosclerotic plaque. MethodsA Cre/mloxP conditional KO system and Tek-Cre mice were used to generate offsprings with monoallelic deletion of the TFPI-2 gene in endothelial cells. TFPI-2fl/+/Tek-Cre mice, TFPI-2fl/+ mice and ApoE−/− mice (n=6 for each group) were included. Arteries were obtained. HE, EVG and anti-α-SMA staining were used to examine the morphology of vessel and plaque. Protein expression and phosphorylation were detected by Western blot or immunohistochemistry. ResultsTFPI-2fl/+/Tek-Cre mice were generated. TFPI-2 level decreased to 40.68% in TFPI-2fl/+/Tek-Cre group. TFPI-2fl/+/Tek-Cre developed plaques when no plaque was found in TFPI-2fl/+ mice. Compared with ApoE−/− group, TFPI-2fl/+/Tek-Cre group has smaller plaque area, decreased lipid content and less buried fibrous cap layers. MMP-2 and MMP-9 in TFPI-2fl/+/Tek-Cre group was higher than in TFPI-2fl/+group. The phosphorylation of PPAR-α and PPAR-γ was decreased in TFPI-2fl/+/Tek-Cre group. ConclusionsA novel mouse model is presented and can be used to investigate the role of TFPI-2 in the process of atherosclerosis. Our findings suggest that monoallelic deletion of TFPI-2 gene in vascular endothelial cells leads to significant downregulation of TFPI-2. TFPI-2 deficiency may accelerate initiation of atherosclerotic lesion in mice. Elevated MMP-2 and 9 and decreased phosphorylation of PPAR-α and PPAR-γ may contribute to this phenotype.

33 Developing a transgenic mouse model with inducible cardiac fibroblast-selective deletion of the interleukin-1 receptor, IL1R1

Rationale Interleukin-1a (IL-1a) released from necrotic cardiomyocytes is hypothesised to stimulate inflammatory responses in cardiac fibroblasts (CF) and contribute to inflammation post-myocardial infarction. Here we describe a murine model with tamoxifen-inducible fibroblast-specific deletion of the IL-1 receptor (IL1R1) for assessing the importance of the IL-1/CF axis in vivo . Methods IL1R1-/- mice and IL1R1flx/flx mice, co-expressing tamoxifen-inducible Cre-recombinase under the control of a fibroblast-specific promoter (Col1a2-CreER(T)), were crossed to produce hemizygous offspring (IL1R1flx/-). Cre-positive IL1R1flx/- (experimental) and Cre-negative IL1R1flx/- (control) mice aged 2–3 weeks were injected with tamoxifen (100mg/kg, 5 days) and hearts isolated 4–8 weeks later to establish primary CF cultures. The functional effect of IL1R1 deletion was determined by measuring cytokine and MMP expression (qRT-PCR, ELISA) in response to IL-1a or TNFa. Results Without tamoxifen administration, IL-1a responses in IL1R1flx/flx and IL1R1flx/- CF were identical despite 50% reduction of IL1R1 mRNA in IL1R1flx/- cells. IL1R1-/- CF showed no response to IL-1a. Monoallelic deletion of IL1R1 therefore had no functional impact. With tamoxifen treatment, a further 75% reduction in basal IL1R1 mRNA levels was observed in Cre-positive IL1R1flx/- CF vs. Cre-negative cells. IL-1a-induced MMP-3, MMP-9 and IL-6 mRNA was reduced by 65–75% in CF from tamoxifen-injected Cre-positive IL1R1flx/- mice vs. control. Similar results were obtained for protein secretion. TNFa responses were unaffected by IL1R1 deletion. Conclusions We have developed a fibroblast-selective IL1R1 knockout mouse in which functional deletion of IL1R1 can be induced in ~75% of CF. The impact of this will be studied in an experimental myocardial infarction model.

Targeting autophagy in cancer management - strategies and developments.

Autophagy is a highly regulated catabolic process involving lysosomal degradation of intracellular components, damaged organelles, misfolded proteins, and toxic aggregates, reducing oxidative stress and protecting cells from damage. The process is also induced in response to various conditions, including nutrient deprivation, metabolic stress, hypoxia, anticancer therapeutics, and radiation therapy to adapt cellular conditions for survival. Autophagy can function as a tumor suppressor mechanism in normal cells and dysregulation of this process (ie, monoallelic Beclin-1 deletion) may lead to malignant transformation and carcinogenesis. In tumors, autophagy is thought to promote tumor growth and progression by helping cells to adapt and survive in metabolically-challenged and harsh tumor microenvironments (ie, hypoxia and acidity). Recent in vitro and in vivo studies in preclinical models suggested that modulation of autophagy can be used as a therapeutic modality to enhance the efficacy of conventional therapies, including chemo and radiation therapy. Currently, more than 30 clinical trials are investigating the effects of autophagy inhibition in combination with cytotoxic chemotherapies and targeted agents in various cancers. In this review, we will discuss the role, molecular mechanism, and regulation of autophagy, while targeting this process as a novel therapeutic modality, in various cancers.

Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma.

BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that is involved in the regulation of cell growth. Recently, many somatic and germline mutations of BAP1 have been reported in a broad spectrum of tumors. In this study, we identified a novel somatic non-synonymous BAP1 mutation, a phenylalanine-to-isoleucine substitution at codon 170 (F170I), in 1 of 49 patients with esophageal squamous cell carcinoma (ESCC). Multiplex ligation-dependent probe amplification (MLPA) of BAP1 gene in this ESCC tumor disclosed monoallelic deletion (LOH), suggesting BAP1 alterations on both alleles in this tumor. The deubiquitinase activity and the auto-deubiquitinase activity of F170I-mutant BAP1 were markedly suppressed compared with wild-type BAP1. In addition, wild-type BAP1 mostly localizes to the nucleus, whereas the F170I mutant preferentially localized in the cytoplasm. Microarray analysis revealed that expression of the F170I mutant drastically altered gene expression profiles compared with expressed wild-type BAP1. Gene-ontology analyses indicated that the F170I mutation altered the expression of genes involved in oncogenic pathways. We found that one candidate, TCEAL7, previously reported as a putative tumor suppressor gene, was significantly induced by wild-type BAP1 as compared to F170I mutant BAP1. Furthermore, we found that the level of BAP1 expression in the nucleus was reduced in 44% of ESCC examined by immunohistochemistry (IHC). Because the nuclear localization of BAP1 is important for its tumor suppressor function, BAP1 may be functionally inactivated in a substantial portion of ESCC. Taken together, BAP1 is likely to function as a tumor suppressor in at least a part of ESCC.