scholarly journals Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis

2019 ◽  
Author(s):  
Samantha R. Soncini ◽  
Dakshayini G. Chandrashekarappa ◽  
David A. Augustine ◽  
Kenny P. Callahan ◽  
Allyson F. O’Donnell ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Tumor Cells ◽  
Genome Sequencing ◽  
Human Tumor ◽  
Whole Genome ◽  
Spontaneous Mutations ◽  
Human Tumor Cells ◽  
Snf1 Kinase ◽  
Fast Growing ◽  
Highly Sensitive

AbstractYeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of the HXK2, REG1, GLC7 and SNF1 genes that were shown to confer significant resistance to 2-deoxyglucose. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1, GLC7 and SNF1) exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose. Deletion of the HXK2 and REG1 genes confers resistance to 2-deoxyglucose and causes increased expression of the DOG2 mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation of DOG2 expression are important mechanisms for resistance to 2-deoxyglucose. However, the dominant SNF1-G53R allele can confer additional 2-deoxyglucose resistance in cells that are genetically compromised in both the endocytosis and DOG pathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.Author SummaryYeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers’ yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.

scholarly journals Draft Genome Sequence of a Mycobacterium porcinum Strain Isolated from a Pet Cat with Atypical Mycobacterial Panniculitis

2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Anthony Mannion ◽  
Tina McCollester ◽  
Alexander Sheh ◽  
Zeli Shen ◽  
Hilda Holcombe ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Whole Genome ◽  
Content Type ◽  
Fast Growing

A fast-growing Mycobacterium species was cultured from draining, purulent lesions on the caudal abdomen of a 12-year-old male domestic long-haired cat. Whole-genome sequencing identified the organism as Mycobacterium porcinum.

Device for whole genome sequencing single circulating tumor cells from whole blood

Lab on a Chip ◽  
2019 ◽  
Vol 19 (19) ◽  
pp. 3168-3178 ◽  
Author(s):  
Ren Li ◽  
Fei Jia ◽  
Weikai Zhang ◽  
Fanghao Shi ◽  
Zhiguo Fang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Genome Sequencing ◽  
Microfluidic Chip ◽  
Whole Blood ◽  
Whole Genome

To sequence single circulating tumor cells (CTCs) from whole blood, a microfluidic chip was developed to perform blood filtering/CTC enrichment/CTC sorting and in situ MDA for whole genome sequencing.

Whole Genome Sequencing Reveals a Widely Expressed Mutation (MYD88 L265P) with Oncogenic Activity in Waldenstrom's Macroglobulinemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 300-300 ◽  
Author(s):  
Steven P Treon ◽  
Lian Xu ◽  
Yangsheng Zhou ◽  
Xia Liu ◽  
Guang Yang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Tumor Cells ◽  
Genome Sequencing ◽  
Sanger Sequencing ◽  
Whole Genome ◽  
Waldenström’S Macroglobulinemia ◽  
Oncogenic Activity ◽  
Waldenstrom's Macroglobulinemia ◽  
Waldenström's Macroglobulinemia ◽  
Myd88 L265p

Abstract Abstract 300 We performed whole genome sequencing (WGS) of lymphoplasmacytic cells from 30 Waldenstrom's Macroglobulinemia (WM) patients, with paired normal tissue sequencing for 10 patients. Tumor and normal genomes were both sequenced to an average of 66X coverage of mapped individual reads. A recurring sequence variant at position 38182641 in chromosome 3p22.2 was identified which resulted in a single nucleotide change from T®C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P). This variant was the most common of a median of 3,419 (range 2,540–4,011) somatic variants identified by WGS in paired patients, and was present in tumor cells from all 10 paired patients, and 16 of 20 unpaired patients. For 22 of 26 patients, the MYD88 L265P variant was heterozygous, whereas in 4 patients an acquired UPD event at 3p22.2 resulted in homozygous presence of the variant in at least a subset of tumor cells. Sanger sequencing confirmed the presence of the MYD88 L265P variant in all 26 patient tumor samples revealed by WGS, as well as in one additional patient's tumor sample that was not identified by the variant calling algorithms but for whom 12% of the WGS read level mappings showed the MYD88 L265P variant. In contrast, the MYD88 L265P variant was absent in normal paired tissues. Sanger sequencing therefore confirmed the somatic presence of the MYD88 L265P variant in tumor cells from 27 of 30 (90%) WM patients, and also identified this variant in BCWM.1 and MWCL-1 WM cells. In contrast, the MYD88 L265P variant was absent in CD138+ selected tumor cells from 8 of 8 multiple myeloma (MM) patients, and CD19+ cells from 12 of 12 healthy individuals, as well as in 7 of 8 patients with IgM monoclonal gammopathy of unknown significance (MGUS), in whom absence of the MYD88 L265P variant was further confirmed by TA cloning and sequencing of at least 100 clones. In the sole IgM MGUS patient in whom the MYD88 L265P mutation was detected, subsequent disease evolution occurred. Importantly, knock-down of MYD88 expression by lentiviral transduction led to loss of NF-κβ signaling and apoptosis of both BCWM.1 and MWCL-1 WM cells, with enhanced survival observed by complementary transduction with MYD88 L265P versus MYD88 wild type. The results of these studies therefore demonstrate a widely expressed somatic variant (MYD88 L265P) in malignant LPC of WM, whose presence confers oncogenic activity, and which may help distinguish WM disease from IgM MGUS or MM. Disclosures: No relevant conflicts of interest to declare.

Abstract 938: Single-cell whole-genome sequencing verifies the surrogacy of circulating tumor cells for prostate cancer

2014 ◽  
Author(s):  
Yi-Tsung Lu ◽  
Runze Jiang ◽  
Hsian-Rong Tseng ◽  
Leland W. Chung ◽  
Edwin M. Posadas
Keyword(s):  
Prostate Cancer ◽  
Whole Genome Sequencing ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Genome Sequencing ◽  
Whole Genome

Prevalence and tissue concordance of BRCA2 copy number loss evaluated by single-cell, shallow whole genome sequencing of circulating tumor cells (CTCs) in castration-resistant prostate cancer (CRPC).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 5531-5531
Author(s):  
Ethan Barnett ◽  
Joseph Schonhoft ◽  
Nikolaus D. Schultz ◽  
Jerry Lee ◽  
Samir Zaidi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Whole Genome Sequencing ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Genome Sequencing ◽  
Copy Number ◽  
Cellular Heterogeneity ◽  
Cancer Tissue ◽  
Whole Genome

5531 Background: Genomic studies have shown that up to 25% of prostate cancer tissue specimens harbor alterations in DNA Damage Repair (DDR) genes, which may sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Trials evaluating PARPi in patients with DDR deficiencies have shown varied response rates and differences regarding which genomic alterations predict for sensitivity to these agents, with the majority of objective responses seen in BRCA2-altered tumors. These results highlight the need to develop biomarker assays which can predict benefit from PARPi therapy. Tissue and cell-free DNA (cfDNA) have been the most utilized sources of tumor material for analysis in this setting, but success rates of obtaining sufficient tumor for analysis from bone are low and detecting tumor-derived copy number variants (CNVs) in cfDNA is challenging. Circulating tumor cells (CTCs) represent an alternate source of genetic information, for which assays are available to isolate and sequence individual cells in a manner that eliminates background noise from stroma and healthy cells, while capturing inter-cellular heterogeneity. Methods: Blood samples, collected from 138 progressing metastatic CRPC patients within 30 days of a pre-treatment biopsy intended for sequencing using MSK-IMPACT, were sent to EPIC Sciences for CTC analysis. Detected CTCs underwent single cell, low pass whole genome sequencing. Prevalence and concordance of BRCA2 copy-loss, regardless of whether single copy or homozygous, was compared in matched tissue and CTC samples. Results: BRCA2 copy-loss was identified in 21% (23/108) and 50% (58/115) of successfully sequenced tissue and CTC samples, respectively. In the 58 patients with CTC-detected BRCA2 loss, BRCA2 loss was detected in 36% (220/565) of the sequenced CTCs, representing a median of 46% (range 4-100%) of CTCs found in each individual sample. When both sequencing assays were successful, BRCA2 loss was detected in CTCs in 84% (16/19) of the tissue-positive cases, whereas tissue sequencing detected BRCA2 loss in 35% (16/46) of CTC-positive cases. Conclusions: Data from this study supports the notion that single-cell CTC sequencing can detect BRCA2 copy-loss at a high frequency, including cases that were negative in tissue, while also characterizing inter-cellular heterogeneity. Further studies will investigate whether CTC BRCA2 copy-loss can predict the likelihood of response to PARPi.

Tumor-targeted oncolytic adenovirus demonstrates high cytotoxicity for human lung and renal cell carcinomas independently of the level of tumor PD-L1 expression.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3596-3596
Author(s):  
Jia Yao ◽  
Svetlana Atasheva ◽  
Cedrick Young ◽  
Viraj A. Master ◽  
Dmitry Shayakhmetov
Keyword(s):  
Virus Infection ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Cell Lines ◽  
Oncolytic Virus ◽  
Human Tumor ◽  
Oncolytic Adenovirus ◽  
Human Tumor Cells ◽  
Highly Sensitive ◽  
Clinical Benefits

3596 Background: Immuno-checkpoint (IC) inhibitors targeting PD1-PD-L1 pathway have proven highly effective at extending survival of cancer patients. However, the clinical benefits of IC inhibitors are limited to only about 20% patients who have moderate to high levels of tumor PD1 and/or PD-L1 expression. To develop therapeutics that would provide clinical benefits to a larger proportion of cancer patients, we engineered oncolytic adenovirus for targeted infection of human tumor cells via CD46 and integrins of α3β1 or α6β4 classes, overexpressed on many epithelial human cancers. Methods: Here, we analyzed the infectivity and cytotoxicity of this novel oncolytic virus in a panel of human non-small cell lung cancers (NCSLC) cell lines, primary patient derived NSCLC xenografts, and tumor surgical explants from patients with renal cell carcinoma (RCC). Results: The in vitro analysis of NSCLC cells lines (N = 17) demonstrated that over 60% of them were highly sensitive to virus infection. The genome-wide transcriptional profiling showed that only 3 out of 12 cell lines that were sensitive to oncolytic virus infection, expressed PD-L1 ( > 4.5 Log2 RPMK). Furthermore, although the pre-treatment of these cell lines with IFN-I activated PD-L1 expression, IFN-I treatment did not reduce the efficacy of tumor cell infection by the oncolytic virus. The analysis of virus infectivity on primary human tumor cells from patients with NSCLC (N = 4) and RCC (N-24) demonstrated that primary tumors were highly sensitive to oncolytic virus infection. Specifically, tumor-targeted oncolytic virus demonstrated strong cytotoxicity in 22 out of 24 analyzed primary isolated RCC cell samples. Next, we subcutaneously grafted PD-L1-negative NSCLC A549 cells to NSG mice, treated them with oncolytic virus intravenously, and the kinetics of tumor growth and animal survival was monitored. This analysis showed that after intravenous administration, oncolytic virus was able to infect tumor cells and suppress tumor growth. Whereas the median survival in mock-treated group was 26 days, all mice survived up to 100 days post oncolytic virus therapy (endpoint). Conclusions: Our study showed that tumor-targeted oncolytic adenovirus infects human tumor cell lines independently of their PD-L1 expression status and is not sensitive to IFN-I inhibition. This novel tumor-targeted oncolytic virus has the potential to provide clinical benefits to cancer patients, who do not respond or became resistant to ICI drugs.

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