Citations

Abstract IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33–mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an ∼3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.

Abstract CLL is a clinically heterogeneous disease with wide ranging disease course. A novel CLL-IPI based on Rai/Binet stage, IGHV-mutation status, TP53 mutation/deletion, B2M level, and age was developed to stratify patients into 4 risk groups, with a c-statistic of 0.75. Next-generation sequencing has identified ~60 genes recurrently mutated in CLL, some of which are associated with poor overall survival, whereas the clinical effect of most genes is still unknown. Herein, we examine whether somatic mutations in these putative driver genes are associated with time to first treatment (TTT), and whether they add prognostic value beyond CLL-IPI. Based on the 2008 International Workshop CLL criteria, we identified 100 CLL and 96 high-count monoclonal B-cell lymphocytosis (MBL) newly diagnosed from the Mayo Clinic CLL biobank. Pre-treatment peripheral blood mononuclear cells were collected <2 years of diagnosis and tumor DNA was extracted from sorted CD5+/CD19+. We sequenced the coding regions of 61 recurrently mutated CLL driver genes using a custom SureSelect panel, with 24 samples per flow cell in Illumina HiSeq 4000. The average coverage depth was >1000X. Somatic mutations were called using MuTect2 in tumor-only mode. To remove germline variants, variants were eliminated based on minor allele frequencies >0.01%, identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering, high/moderate impact mutations were analyzed using Cox regression, to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT. Among 196 patients the most commonly mutated genes were TP53 (11%), ATM (10%), SF3B1 (10%), NOTCH1 (9%), CHD2 (8%), and BIRC3 (7%). The median follow-up was 8.7 years, and 73 patients were subsequently treated. ATM (HR=3.27, CI:1.8-6.1, P=0.0002) and NOTCH1 (HR=2.41, CI:1.3-4.6, P=0.008) were associated with TTT. When evaluating the total number of mutated genes, we found 32%, 29%, and 39% patients had ≥2, 1, or 0 genes mutated, respectively, and this was associated with shorter TTT (HR=1.74, CI:1.3-2.4, P=0.0005) adjusting for sex and CLL-IPI with a c-statistic=0.8 (CI: 0.75-0.84). When stratified by CLL-IPI, the association held for low (N=99, HR=1.88, CI:1.1-3.4, P=0.03) and intermediate risk (N=54, HR=1.87, CI:1.1-3.2, P=0.03) but not high/very high risk (N=35, HR=1.07, CI:0.6-1.9, P=0.83). We demonstrated that the total number of CLL putative driver genes with high or moderate impact mutations provided prognostic information in newly diagnosed CLL/MBL beyond CLL-IPI. Moreover, even among those with low or intermediate CLL-IPI risk, the total number of somatic mutations separated those patients who progressed. Sequencing the CLL driver genes at time of diagnosis could be a potential biomarker for outcome prediction. Citation Format: Geffen Kleinstern, Daniel R. O’Brien, Brian F. Kabat, Kari G. Chaffee, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Somatic mutations within chronic lymphocytic leukemia (CLL) putative driver genes are associated with outcomes beyond the CLL international prognostic index (CLL-IPI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4466.

Abstract CLL is a highly heritable cancer with first degree relatives of CLL cases having a 7.5-fold increased CLL risk. Genome-wide association studies (GWAS) and linkage studies have been performed to study inherited predisposition; however a larger proportion of heritability to CLL remains unexplained. Rare coding variants might account for the missing heritability information. Inherited loss of function variants in shelterin complex genes (POT1, ACD, TERF1, TINF2, TERF2, TERF2IP- involved in telomere regulation), CDK1 (critical for cell division) and ATM (tumor suppressor gene) have been found to co-segregate in CLL families and be enriched in CLL cases using exome-wide sequencing data. Our study evaluates rare germline variants from these suspect genes segregating in CLL families who are followed at the Mayo Clinic. Using whole exome sequencing (WES), we sequenced 93 CLL families with at least 2 reported CLL cases consisting of 443 individuals: 160 with CLL, 73 with monoclonal B-cell lymphocytosis (MBL), and 210 relatives. DNA was extracted from buccal cells, coding exons were selectively captured using Agilent 50Mb and SureSelect Human All Exon V4 capture kits; sequencing was performed using Illumina HiSeq 2000. Mayo Clinic's DNASeq pipeline uses Novoalign (initial read alignment), Picard (marking duplicate reads), and the Genome Analysis Toolkit (GATK) for local realignment, recalibration, and variant calling. The variant discovery step leverages GATK's HaplotypeCaller in per sample mode and all of the samples across the cohort are jointly genotyped together. All called variants are evaluated with GATK's Variant Quality Score Recalibration tool and annotated for biological relevance (BIOR). Quality control included removing variants that had <75% call rate across the two capture kits, <8x coverage, or phred score<10, resulting in 317,666 remaining variants. Of these, over 80% of the coding sequence had a median read depth of 23 reads. In our pedigrees, we searched for rare variants within the genes described above. We identified suspect variants with the following criteria: 1) enriched in CLL and MBL samples compared to unaffected samples; 2) multiple affected members with the variant within a family; 3) variants present in all sequenced affecteds within the family; 4) rarely seen in an in-house database of non-cancer controls or 1K Genomes; and 5) predicted to have a functional damaging effect (using SIFT). We identified three novel rare missense variants, defined as functionally deleterious, which each co-segregated within a CLL family. Specifically, these variants from shelterin complex genes; POT1 (rs116916706), TERF2IP (rs138458227), and TERF2 (rs749171225), met the criteria. This study further highlights telomere dysregulation as a key process in CLL development. Investigating rare variants within CLL pedigrees with WES can help identify germline variants impacting predisposition to familial CLL. Citation Format: Alyssa I. Clay-Gilmour, Daniel R. O'Brien, Sara J. Achenbach, Celine M. Vachon, Kari G. Chaffee, Timothy G. Call, Jose F. Leis, Aaron D. Norman, Brian F. Kabat, Sameer A. Parikh, Neil E. Kay, Esteban Braggio, James R. Cerhan, Susan L. Slager. Rare germline variants segregating in chronic lymphocytic leukemia (CLL) families [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1226.

There is no therapy that promotes maturation and functionality of a dialysis arteriovenous fistula (AVF). The search for such therapies largely relies on evaluation of vascular responses and putative therapies in experimental AVFs. We studied an AVF in mice with chronic kidney disease (CKD). We demonstrate numerous stressors in the vein of the AVF-CKD group, including pathological shear, mitogenic, inflammatory, and hypoxia-reoxygenation stress. Because stress promotes premature senescence, we examined whether senescence is induced in the vein of the AVF-CKD model. We demonstrate a senescence phenotype in the AVF-CKD model, as indicated by increased expression of p16Ink4a, p21Cip1, and p53 and expected changes for certain senescence-associated microRNAs. RNA-sequencing analysis demonstrated differential expression of ~10,000 genes, including upregulation of proinflammatory and proliferative genes, in the vein of the AVF-CKD group. The vein in the AVF-CKD group exhibited telomere erosion and increased senescence-associated β-galactosidase activity and staining. Senescence was induced in the artery of the AVF-CKD group and in the vein of the AVF without CKD. Finally, given the rapidly rising clinical interest in senolytics, we provide proof of concept of senolytics as a therapeutic approach by demonstrating that senolytics decrease p16Ink4a expression in the AVF-CKD model. This study introduces a novel concept underlying the basis for maturational and functional failure in human dialysis AVFs and identifies a new target for senolytic therapy.

Chromosome instability (CIN) is widely observed in both sporadic and hereditary colorectal cancer (CRC). Defects in APC and WNT signaling are primarily associated with CIN in hereditary CRC, but the genetic causes for CIN in sporadic CRC remain elusive. Using high‐density SNP array and exome data from The Cancer Genome Atlas (TCGA), we characterized loss of heterozygosity (LOH) and copy number variation (CNV) in the peripheral blood, normal colon, and corresponding tumor tissue in 15 CRC patients with proficient mismatch repair (MMR) and 24 CRC patients with deficient MMR. We found a high frequency of 18q LOH in tumors and arm‐specific enrichment of genetic aberrations on 18q in the normal colon (primarily copy neutral LOH) and blood (primarily copy gain). These aberrations were specific to the sporadic, pMMR CRC. Though in tumor samples genetic aberrations were observed for genes commonly mutated in hereditary CRC (eg, APC, CTNNB1, SMAD4, BRAF), none of them showed LOH or CNV in the normal colon or blood. DCC located on 18q21.1 topped the list of genes with genetic aberrations in the tumor. In an independent cohort of 13 patients subjected to Whole Genome Sequencing (WGS), we found LOH and CNV on 18q in adenomatous polyp and tumor tissues. Our data suggests that patients with sporadic CRC may have genetic aberrations preferentially enriched on 18q in their blood, normal colon epithelium, and non‐malignant polyp lesions that may prove useful as a clinical marker for sporadic CRC detection and risk assessment.

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (T CM ) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected T CM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo . Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV + memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

ABSTRACT We report on nine draft genomes of Pseudomonas aeruginosa isolates, assembled using a hybrid paired-end and Nextera mate-pair library approach. Eight are of clinical origin, and one is the ATCC 27853 strain. We also report their multilocus sequence types.