Vivien Hauer

Bio: Vivien Hauer is an academic researcher from Leibniz Association. The author has contributed to research in topics: Lymphoma & Apoptosis. The author has an hindex of 3, co-authored 7 publications receiving 21 citations.

Identification of cell lines CL-14, CL-40 and CAL-51 as suitable models for SARS-CoV-2 infection studies.

Abstract: The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.

RBFOX2 and Alternative Splicing in B-cell Lymphoma

Abstract: RBFOX2 is a master regulator of alternative splicing. This RNA-binding protein (RBP) is expressed in the brain, muscle, and embryonic stem cells. RBFOX2 is required not only for the proper splicing of target RNAs, but also for cerebellar development, myogenesis, and for survival of human embryonic stem cells. Hitherto, not much is known about the expression and function of RBFOX2 in hematopoetic tissues. In an early report, RBFOX2 had been shown to be capable of promoting inclusion of exon16 in protein 4.1R. This splicing event is important for erythropoiesis because it increases the affinity of 4.1R for target genes. In a recent study, expression of RBFOX2 was detected in the human T-cell line JURKAT and a functional antagonism of the RBPs RBFOX2 and CELF2 was demonstrated. We wanted to describe the expression patterns of RBFOX2 in hematopoetic malignancies, to discover target genes and to unravel the consequence of RBFOX2 repression for target gene splicing and isoform expression. Expression array and Western blot analysis showed that human B non-Hodgkin lymphoma (B-NHL) cell lines are RBFOX2 negative or positive (Supplement 1A). To find the potential targets of the splice factor RBFOX2, we compared the expression of individual exons in RBFOX2negative and RBFOX2-positive cell lines. This approach relied on the assumption that the differential expression of this RBP would provoke changes in the expression of individual exons and would thereby allow identification of target genes. Supplement 1B shortlists these genes ordered by statistical significance. Expression of the individual exons and joining sequences of MALT1 is shown as heatmap in Fig. 1a. The full-length MALT1 variant was associated with RBFOX2 expression (Fig. 1a). Results of splice variant analysis with a larger panel of cell lines revealed a striking association between expression of RBFOX2 and expression of the full-length forms of all four candidate target genes,MALT1, CLSTN1, FMNL3, and MYO9B (Fig. 1b). The short variants were expressed in RBFOX2-negative cell lines only (Fig. 1b). Two of these potential RBFOX2 target genes (CLSTN1 and FMNL3) had already been described in the context of RBFOX2mediated splicing. The RBFOX2 target sequence “UGCAUG” was present in all introns following the retained exons, indicating that high RBFOX2 levels might be the cause of the full-length forms in the RBFOX2 positive cell lines. Supporting the notion that RBFOX2 was important for splicing of these genes was also the finding that RBFOX2 was the sole gene that was significantly overexpressed in cell lines expressing full-length MALT1 when compared to cell lines expressing MALT1 without exon 7 (Supplement 1C). We performed knockdown experiments to test whether RBFOX2 was responsible for retaining MALT1 exon 7, FMNL3 exon 26, and MYO9B exon 37. siRNAs reduced expression of RBFOX2 in RBFOX2-positive cell lines BL2, SU-DHL-5, and HT by more than 50% (Fig. 1c, Supplement 1D). Repression of RBFOX2 induced the shorter isoforms of MALT1 (w/o exon 7), MYO9B (w/o exon 37), and FMNL3 (w/o exon 26) (Fig. 1c, Supplement 1D). The long form of CLSTN1, the fourth gene tested here, was not or only marginally expressed in BL-2, SU-DHL-5, and HT cells, explaining why we could not observe an increase of the short isoform of this gene after RBFOX2 knockdown (data not shown). In sum, our data showed that

Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing

Abstract: Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.

Chromosome 11q23 aberrations activating FOXR1 in B-cell lymphoma.

Abstract: Recurrent chromosome 11q23 abnormalities, including focal gains and losses have been described in mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL) and in a subset of high-grade B-cell lymphomas lacking MYC rearrangements. We describe a novel fusion of FOXR1 forkhead box gene, located at 11q23, with a neighboring gene in B-cell lymphoma. RNAseq and sequencing of cloned PCR products revealed fusion transcripts of 5′ Ribosomal Protein S25 (RPS25) with FOXR1 in the DLBCL cell line U-2932, both genes located at the amplified chromosomal region 11q23 (Figure 1a, Supplementary Figure S1A). Genomic cloning localized the breakpoint to intron 2/3 of RPS25 and to the promoter region of FOXR1 (bp − 3532). Cell line U-2932 comprises two distinct clones traceable to subclones present in the patient ́s tumor. These differences also affected 11q32, FOXR1 and RPS25 being tetraploid in subclone R1, and triploid in subclone R2 (3n) (Supplementary Figure S1A). In accordance with the genomic data, the RPS25/FOXR1 fusion was detected in subclone R1 but not in R2 (Figure 1b). RPS25/FOXR1 was also verified in the patient ́s DNA, which collectively suggested that the fusion had occurred at some later stages of tumor development (Figure 1b). Physiological FOXR1 (formerly FOXN5) expression is restricted to the early stages of embryogenesis. Ectopic expression as result of 11q23 intrachromosomal deletion-fusion has hitherto been described in neuroblastoma only. In-frame fusions with the 5′ MLL or PAFAH1B2 genes led to overexpression of FOXR1. In accordance with the notion that a constitutively expressed 5′ gene (RPS25) might be responsible for the ectopic expression of FOXR1 in B-cell lymphoma also, FOXR1 levels were 1000 × higher in the fusion-positive than in the fusion-negative U-2932 subclone. Expression array analyses showed that the RPS25/FOXR1-positive U-2932 subclone had the highest FOXR1 expression level of 55 B lymphoma cell lines tested, three log-scales higher than average (Figure 2a). Quantitative PCR analysis conducted to verify the expression arrays included 17 additional B lymphoma cell lines, revealing that the primary effusion lymphoma cell line CRO-AP3

Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

Abstract: Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

Abstract: The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

Abstract: The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia

Abstract: Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.

Inactivating mutations of the histone methyltransferase EZH2 in myeloid disorders

Abstract: Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

SARS-CoV-2 Omicron spike mediated immune escape and tropism shift

Abstract: The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.