Search

Genomic profiling of glioblastoma uncovers fundamental differences in the driver events of malignant transformation and progression, yet the nuances driving the particular growth rate of glioblastoma in individual patients remain poorly defined. We exploited the availability of fourteen histocompatibility-matched mouse hosts from the collaborative cross (CC) [PMID: 15514660] as hosts for a non-germline, engineered model of murine glioblastoma [PMCID: PMC4210043] to interrogate cell types, functional states and proximity in situ in tumors with varying growth rates. Specifically, Kaplan-Meier survival data was used to determine the rates of Nf1:TP53 null tumor growth in different cc host groups. Spatial transcriptomics and single nucleus RNA seq (sn-seq) analyses were conducted in tumor-bearing brain tissue from C57Bl/6 (syngeneic) and 2 CC histocompatible allogeneic mouse strains with differential survival outcomes. Spatial analysis was conducted using a disease-focused multiplexed mRNA probe set assembled from annotations of GBM/glioma specific genes reported in the literature. Cell segmentation of the data was performed to investigate the spatial pattern of single-cell level expression. The spatial transcriptomic dataset was also analyzed with a segmentation-free pipeline. In addition, to detect cell clusters, the probability of genes detected in close proximity (genes within a cell 5px radius [0.5 um]) was determined using Pdgfra as the tumor marker (anchor). We identified differential levels of spatially correlated expression of cell clusters expressing hypoxia markers such as HIF-1a and PDK1 between hosts with fast and slow growing tumors. Tumors from the 3 hosts were also processed for sn-seq. Integrated analysis of cell clustering was used to determine differences in clusters between the fast growing and slow growing groups. Enrichment of expression of Top2a, a gene associated with proliferation and poor prognosis in glioma, was detected in a unique cluster of cells in the slow-growing tumor when compared to the fast-growing group. This suggests that the host microenvironment may play a role in influencing the decelerated growth of this tumor. These results support the utility of spatial transcriptomics and sn-seq in understanding how differences in tumor:host cell interactions contribute to tumor growth. Future opportunities enabled by histocompatible allogeneic hosts for GEM and nGEM glioma models include unraveling host effects on drug sensitivity, tumor heterogeneity, molecular plasticity, angiogenesis, invasion, and lymphoid and myeloid microenvironmental dependencies, each with profound opportunities for clinical translation. Citation Format: Angela S. Baker. Orthotopic nGEM glioma growth rates in allogenic hosts associate with hypoxia and proximity to host cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 10.

Purpose: Prostate cancer is the most common cancer in males, but also exhibits a ∼1.5-2-fold higher incidence in African American men compared with whites. Epidemiologic evidence supports a large heritable contribution to prostate cancer, with over 100 susceptibility loci identified to date that can explain ∼33 % of the familial risk. A portion of the undefined risk may be due to rare susceptibility variants. The African American Hereditary Prostate Cancer (AAHPC) Study, established in 1997, enrolled 77 AA families from seven clinical sites across the United States (Ann Epidemiol, 2000). The aim of this study is to identify rare, predictive, deleterious variants through exome sequencing of 99 cases from 26 families selected from the AAHPC families (2 to 3 affected men sequenced per family) and three female 1000 Genome controls. Methods: To explore the contribution of rare variation in coding regions of 38 known cancer-causing genes to prostate cancer risk (PCa), we sequenced the exomes of 99 AAHPC cases at a mean coverage of 30x. Post-variant calling quality control (QC) was implemented using Golden Helix SVS 8 software with filters set for removal of variants with Read Depth >10, Quality Score >10, and Quality Score: Read Depth Ratio > 0.5. Mendelian inconsistency was checked using PLINK. Prioritization of all candidate genes/variants were evaluated using online databases including 1000 Genomes and ANNOVAR for non-reference allele frequency and predictions of functional impact. Conclusions. Through exome sequencing of 99 AAHPC cases and 3 female 1000 Genome controls, we identified 37 non-synonymous single nucleotide variants that are considered damaging by at least one predictive scoring tool in our 38 candidate genes. However, most of these variants were common and thus unlikely to be causal. We observed three rare variants that were considered damaging by three predictive scoring tools in two known PCa genes, PCNT and ZFHX3. These 3 variants were each observed in a single different family (in 1 of 3 affected individuals). Interesting rare candidate variants (MAF ≤ 0.01) rs190517526 & rs114692729 were found in known cancer susceptibility loci (CD82 and EZH2). The CD82 variant was observed in all 3 sequenced affecteds in one family and the EZH2 variant was observed in 2 of 3 sequenced affecteds in a different family. These predicted damaging variants in these four genes represent potentially novel causal candidates for some of the 26 AAHPC families sequenced here. Future work will carefully analyze the remainder of the genome in the other 21 sequenced families including planned sequencing of additional family members. Citation Format: Deyana D. Lewis, Shukmei Wong, Angela S. Baker, Isaac Powell, John D. Carpten, Joan E. Bailey-Wilson, Cheryl Cropp. Rare candidate variants shared among affected family members in the African American Hereditary Prostate Cancer Study families [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 4240.

Supplemental digital content is available in the text., Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.

Lung cancer remains the leading cause of cancer deaths in the US and throughout the world. Never-smokers with lung cancer constitute an understudied subset of these patients, though recent estimates show that ∼10% of men and women with lung cancer in the US are never-smokers. Independently, lung cancer in never-smokers ranks among the ten most common causes of cancer mortality, and thus the causes, driver pathways, and potential therapeutics must be investigated for this clinically relevant subpopulation. We hypothesize that novel mutations and pathways identified by whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) drive tumorigenesis in adenocarcinomas of never-smoker patients, and represent potential therapeutic targets. We have completed WGS and WTS on two lung adenocarcinomas from female, never-smokers, one early-stage and one advanced-stage (metastatic), and one female smoker patient with early-stage lung adenocarcinoma. Approximately 100 short nucleotide variants (SNV) causing non-synonymous DNA sequence changes were discerned from the early- and advanced-stage adenocarcinomas from never-smokers. Of interest, these never-smoker patients lacked alterations in common genes associated with lung cancer such as EGFR, KRAS, and EML/ALK translocations, making them ideal cases for the discovery of new mutations that may drive lung adenocarcinomas in never-smokers. In comparison, the adenocarcinoma from a smoker patient contained 78 SNVs, including a well-characterized KRAS mutation. Mutations in MAGEC1, a tumor marker in melanoma and multiple myeloma, were observed in common between the early-stage, never-smoker tumor and the smoker tumor. The mutations observed in the never-smoker cases included genes suspected to play a role in lung carcinogenesis. The early-stage, never-smoker patient contained a mutation in PIK3C3 and DOCK10, known to play a role in cancer cell migration, and CSNK1E, a casein kinase involved Wnt signaling and in beta catenin-induced cancer cell proliferation. The late-stage never-smoker patient demonstrated a p53 mutation, a mutation in the tumor suppressor LATS2, and a mutation in the DNA damage checkpoint gene, ATM. The mutations discovered in known tumor suppressor genes tended to be in the late-stage, never-smoke patient. Ingenuity Pathway Analysis implicated G-protein coupled receptor signaling for the early-stage never-smoker, Hif1α signaling for the smoker, and Sonic Hedgehog Signaling for the late-stage, never-smoker in both WGS and WTS. Currently, we are validating the identified mutations discovered by WGS using Sanger sequencing and surveying their frequency in ∼30 and ∼60 additional cases of lung adenocarcinoma from never-smokers and clinically matched smokers, respectively. Validation of these mutations and pathways will lead to a better understanding of tumorigenesis and tumor progression in never-smokers and identify nodes for therapeutic vulnerability.