Citations

Abstract Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

Abstract KaLwRij/C57Bl (KaLwRij, “Kal-ridge”) is a spontaneous mutant mouse strain predisposed to monoclonal gammopathy of uncertain significance (MGUS) and parallels many features of human pathology including progression to multiple myeloma (MM). In African Americans, an increased prevalence of MM is due to an increased risk of MGUS development (rather than increased rate of progression from MGUS) and is likely therefore mediated by germline MGUS risk alleles. In both humans and mice, the genetic basis for MGUS/MM risk is unknown. We sought to identify the genetic basis for MGUS/MM risk in KaLwRij mice to shed light on genes or pathways that might mediate risk in humans. We employed a novel integrative computational analysis, combining KaLwRij/B6 modified haplotype block analysis and low-power human MM/control single nucleotide polymorphism (SNP) data. We used high-density SNP arrays to establish genetic distances of KaLwRij from 11 another diverse inbred strains and confirmed close homology to parent C57Bl/6. Haplotype block analysis identified genetic loci that diverged from C57Bl/6 and included over 500 genes. We filtered the haplotype list using previously published RNA expression data from the bone marrow of C57Bl/6 and KaLwRij mice, to identify 71 differentially expressed genes. As a final filter, we used SNP data from a genome wide association study (GWAS) of human Caucasian MM case and normal volunteer samples (∼300 each) to identify SNPs differentially represented in MM patients. Combining these sets yielded four (4) candidate genes. Our top candidate, Samsn1 (Hacs1), is a negative regulator of B-cell activation and interestingly, was first identified as a gene differentially expressed in MM. Strikingly, we found that KaLwRij mice had a complete germline deletion of the Samsn1 gene. Samsn1 is expressed within the hematopoetic compartment, including B-cells, macrophages, and osteoclasts (OC), key cell types that participate in MM pathology. Consistent with the reported inhibitory function of SAMSN1, KaLwRij had enhanced naïve B-cell response and constitutive Lyn phosphorylation. Further, we found that KaLwRij macrophages and OC also have constitutive Lyn activation consistent activation of cytokine signaling pathways in stromal cells. Absence of Samsn1 may lead to unregulated B-cell and macrophage/OC activation, priming both a pro-tumor bone microenvironment and B-cells for development of MGUS and predisposition to MM. Our approach focused on germline loci rather than malignant cell genetic alterations, advancing our understanding of host predisposition. Identification of the gene or genes predisposing patients to MGUS/MM will provide novel targets for identifying at-risk individuals and will potentially allow the develop of preventative therapeutics for MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract SCI-3 The processes that lead to the susceptibility of post-mitotic tissues to diseases of aging are poorly understood, but mitochondria are considered a key regulator. SIRT3 is mitochondrial sirtuin, a member a family of NAD+-dependent deacetylases. We have previously shown that increased levels of NAD+ in mitochondria promote cell survival in a SIRT3-dependent manner, but how this protection is mediated is unknown. Here we show that mitochondrial NAD+ levels decline dramatically with age and that SIRT3 is a critical component and regulator of the mitochondrial transition pore (MTP). Mice lacking SIRT3 exhibit hyperacetylation of the MTP with age, and as a result, develop multiple defects in post-mitotic tissues as they age, including mitochondrial defects, susceptibility to cardiac failure, and learning and memory deficits. In a mouse model of Alzheimer's disease (AD), SIRT3 knockout mice show striking phenotypes including small size, kyphosis, and a markedly reduced lifespan. Similar to mice, human brain samples show that acetylation of the MTP increases with age and with AD. These findings indicate that declining NAD levels with age lead to memory deficits and accelerate age-related diseases in post-mitotic tissues due to a decline in SIRT3 activity and unscheduled opening of the MTP. Disclosures: Sinclair: GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Abstract Background: Retinoblastoma (RB) is the most frequent pediatric retinal tumor. In the present study, to elucidate chemoresistance mechanisms and identify potential biomarkers in RB, we utilized RNA sequencing (RNAseq) technological platforms to reveal transcriptome profiles and identify any differentially expressed genes (DEGs) between an etoposide drug-resistant subline (Y79/EDR) and parental Y79 cells. Methods: To test whether Y79/EDR cells showed resistance to antineoplastic agents for RB, we treated the cells with etoposide, carboplatin and vincristine and analyzed them with a Cell Counting Kit-8 (CCK-8). Y79/EDR and parental Y79 cells were used for RNAseq and bioinformatics analysis to enable a genome-wide review of DEGs between the two lines using the DESeq R package (1.10.1). Then, DEG enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed with KOBAS software. Next, real-time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR) and cytotoxicity assays were performed to experimentally and functionally validate the identified candidate biomarkers. Results: Y79/EDR cells showed resistance to etoposide, carboplatin and vincristine at different concentrations. In total, 524 transcripts were differentially expressed in Y79/EDR cells based on analysis of fragments per kilobase of transcript per million fragments mapped (FPKM); among these, 57 genes were downregulated and 467 genes were upregulated in Y79/EDR cells compared to parental Y79 cells. We selected candidate DEGs, including ARHGAP9 , HIST1H4H , RELN , DDIT4 , HK2 , STC1 and PFKFB4, for mRNA expression validation with real time QRT-PCR assays and found that the expression levels determined by real time QRT-PCR were consistent with the RNAseq data. Further studies involving downregulation of ARHGAP9 with a specific siRNA showed that ARHGAP9 altered the cellular sensitivity of Y79 cells to etoposide and carboplatin. Conclusion: Our initial findings provided a genomic view of the transcription profiles of etoposide-induced acquired resistance in RB. Follow-up studies indicated that ARHGAP9 might be a chemoresistance biomarker in RB, providing insight into potential therapeutic targets for overcoming acquired chemoresistance in RB. These findings can aid in understanding and overcoming chemoresistance during treatment of RB in the clinic.

Abstract Multiple myeloma (MM) is a malignancy of plasma B-cells that is invariably preceded by monoclonal gammopathy of undetermined significance (MGUS), a pre-neoplastic plasma cell proliferative disorder. C57Bl/KaLwRij (KaLwRij) is a spontaneous inbred mouse strain predisposed to benign idiopathic paraproteinemia (BIP), a plasma cell dyscrasia that parallels many features of human pathology including progression to myeloma. In both humans and mice, the genetic basis for MGUS/MM risk is unknown. We sought to identify the genetic basis for MGUS/MM risk in KaLwRij mice to shed light on genes or pathways that mediate disease risk in humans. Using an integrative approach, we combined KaLwRij x B6 haplotype analysis (418 genes) and a MM patient association study (180 genes) to identify five candidate genes likely to contribute to both murine BIP-susceptibility and human MM risk. Surprisingly, we found KaLwRij mice had homozygous germline deletion of one of the five candidate genes, Samsn1, a putative negative regulator of B-cell activation. Consistent with the reported inhibitory function of SAMSN1 in B-cells, KaLwRij mice had enhanced naïve B-cell response in vitro following stimulation with IL4 and LPS and in vivo following immunization. We found that re-expression of Samsn1 in KaLwRij-derived myeloma cell line 5TGM1 decreased MM tumor growth in vitro, suggesting that loss of Samsn1 contributes to tumor growth in part through a cell intrinsic mechanism. Importantly, germline risk alleles may also influence cell types beyond the pre-malignant B-cells. It is established that the host environment also plays a critical role in supporting myeloma growth in KaLwRij mice. We found that Samsn1 is highly expressed in macrophages, but not in other MM-supportive cell types including osteoclasts and bone marrow stromal cells. Myeloma-associated macrophages play critical roles in supporting tumor growth and are broadly classified as anti-tumor M1 macrophages and tumor-promoting M2 macrophages. Microarray analysis of macrophages from B6 and KaLwRij mice prior to onset of BIP revealed three differentially expressed genes, but only one, Samsn1, is conserved in the human genome. We found that both unpolararized primary peritoneal and MCSF cultured bone marrow macrophages from healthy KaLwRij mice had markedly elevated expression of pro-tumorigenic M2 macrophage markers FIZZ1 and YM1 compared to B6 controls, despite the absence of activating cytokines. To confirm that loss of Samsn1 influenced macrophage activation and M2 polarization, we obtained bone marrow macrophages from mice with a targeted deletion of Samsn1 and found that unpolarized Samsn1-/- cells had similarly increased pro-tumor M2 macrophage marker expression. To evaluate the biologic significance of Samsn1 loss in macrophages on MM tumor growth, we established 5TGM1 MM tumors and injected M2 polarized macrophages from B6, KaLwRij, or Samsn1-/- mice. We found that MM tumor burden was markedly enhanced by the addition of a Samsn1-/- or KaLwRij macrophages compared to wild type macrophages. Thus, we found that loss of Samsn1 in macrophages enhances M2 polarization, prior to MGUS/MM development and can promote established myeloma tumor growth. Together, this data suggests that Samsn1 is a gene contributing to BIP-susceptibility in KaLwRij mice through mutually exclusive functions in multiple cell types including B-cells and macrophages. It is likely that SAMSN1 and its pathway members contribute to human MM progression, and may represent a point for preventative therapeutic intervention. Disclosures No relevant conflicts of interest to declare.

Abstract Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.

AbstractMounting evidence highlights long non‐coding RNAs (lncRNAs) as crucial regulators in multiple types of biological processes and contributing to tumourigenesis. LINC01133, located in chromosome 1q23.2, was a recently identified novel lncRNA with a length of 1154nt. It was involved in the development of colorectal cancer and non‐small cell lung cancer. However, its clinical relevance, biological functions and potential molecular mechanism in breast cancer are still unclear. In this study, we found that the LINC01133 expression was significantly down‐regulated in breast cancer samples and was associated with progression and poor prognosis of breast cancer. Further experiments demonstrated that overexpression of LINC01133 inhibited invasion and metastasis in breast cancer both in vitro and in vivo. Mechanistic investigations revealed that LINC01133 repressed SOX4 expression by recruiting EZH2 to SOX4 promoter. Moreover, rescue experiments further confirmed that LINC01133 functional acted as an anti‐oncogene, at least partly, via repressing SOX4 in breast cancer. Taken together, these findings imply that LINC01133 could serve as a novel prognostic biomarker and potential therapeutic target for breast cancer.

Abstract Single-stranded DNA (ssDNA) sequencing library method has shown to enrich shorter and degraded DNA fragments than double-stranded DNA (dsDNA) library. The goal of this study is to determine whether ssDNA libraries enriches more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA). We used 2ng cfDNA to prepare dsDNA libraries and ssDNA libraries. We also prepare pure-ssDNA libraries by skipping the DNA denaturation step to capture the pre-existing ssDNA only. To calculate ctDNA content, we mapped fastq files to human genome and binned the mapped reads into 1 Mb genomic windows. We normalized the read counts using two unrelated healthy controls. The resulting ratios were transformed with log2 and adjusted for GC content. The fully normalized log2 ratios were subjected to segmentation using the copy number analysis method. After the segmentation, we selected the most significantly deleted genomic region with segment size >20 Mb in each library pair and calculated ctDNA content using formula: 100 × (1-2log2 ratio). We also calculated the plasma genomic abnormality (PGA) score (a composite score algorithm to reflect overall ctDNA burden in peripheral blood) to estimate ctDNA burden. Overall, we prepared and sequenced a total of 27 libraries including ten dsDNA libraries, ten ssDNA libraries and seven pure-ssDNA libraries, using ten cfDNAs from cancer patients. We received an average of 24264998 raw read sequences and 19495014 mappable reads per sequencing library. Duplicate rate in the ssDNA libraries (mean=0.057%, range=0.046-0.083%) and pure-ssDNA libraries (mean=0.06%, range= 0.054-0.065%) was significantly lower than in dsDNA libraries (mean=0.2%, range=0.14-0.249%) (p<0.001 and p<0.01, respectively), indicating that ssDNA-based library preparation method may better preserve the diversity and complexity of ctDNAs. Library insert size analysis showed that ssDNA libraries were on average 14bp shorter (range from 6 bp to 21 bp) than their matched dsDNA libraries. We also found the Ampure XP beads ratio influenced the library size. We observed consistently higher ctDNA content in ssDNA libraries than in matched dsDNA libraries (p<0.0005). Comparing ssDNA libraries (mean ctDNA content=34%) to their matched dsDNA libraries (mean ctDNA content=30.7%), the increased ctDNA content ranged from 1.3% to 6.4% (mean=3.3%). ctDNA content was also significantly higher in pure-ssDNA libraries than in their matched dsDNA libraries (p<0.005). The increased ctDNA content ranged from 1.0% to 6.9% (mean=3.6%). The PGA scores were always higher in the ssDNA libraries (p<0.0001) or pure-ssDNA libraries (p<0.005) compared to the matched dsDNA method. Our results demonstrate that ssDNA libraries enrich more ctDNA than dsDNA libraries do. Pre-existing ssDNA in plasma is abundant and provides sufficient resource for genomic analysis of ctDNA. Citation Format: Jing Zhu, Jingyong Huang, Peng Zhang, Manish Kohli, Chiang-Ching Huang, Liang Wang. Advantages of ssDNA over dsDNA library preparation for capturing cell-free tumor DNA in plasma [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 3536.