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Item A Mechanistic Approach to Identify Novel Therapeutic Drugs for Targeting FA-Disrupted Malignancies(2023-07) Sheth, Aditya Sukumar; Clapp, D. Wade; Vance, Gail; Angus, Steve; Herbert, Brittney-SheaThe Fanconi anemia (FA) signaling network plays a critical role in maintaining genomic integrity during interphase and mitosis. Biallelic germline mutation of any of the 22 genes that constitute this pathway (FANCA-FANCW) results in Fanconi Anemia, a cancer predisposition syndrome characterized by congenital malformations, bone marrow failure, and pediatric acute myeloid leukemias (AMLs). Among the general population, acquired genetic disruptions of the FA pathway are found in 30% of all sporadic cancers and over 15% of sporadic pediatric AMLs underscoring the importance of this pathway in the prevention of malignant transformation. Therefore, the identification of precision therapies for FA-deficient AML is a critical need. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established. We and others have uncovered the roles of FA proteins in mitotic regulation, suggesting additional mechanisms by which the FA pathway prevents genomic instability. Mutation of FANCA is the most common cause of FA and is one of the most frequently disrupted FA pathway genes in sporadic AML. To identify synthetic lethal targets of FANCA, we previously identified mitotic phospho-signaling pathways required for the survival of FANCA-/- patient-derived fibroblasts through a kinome-wide shRNA screen. We identified mitotic kinases CHEK1, PLK1, SLK, and TTK as potential targets, which suggests a mitosis-specific vulnerability of FA-deficient cells. These findings corroborate work by others who have identified synthetic lethal interactions between PLK1 and the FA pathway members, FANCG and BRCA1, suggesting that inactivation of the FA pathway may sensitize cancers to PLK1 inhibition. A more thorough understanding of FA pathway function in mitosis provides new insight into AML pathogenesis and suggests that genetic disruptions of the FA pathway may be predictive of sensitivity to PLK1 inhibition, providing a preclinical rationale for the development of precision therapies.Item Advancing the Safety of Lentiviral Vector Mediated Gene Therapy(2015-04) Shaw, Aaron Marcus; Cornetta, KennethLentiviral vector mediated gene therapy has made great strides in recent years with several successful clinical trials. However, adverse events encountered with some early trials have highlighted the necessity to improve upon its safety. Improvements can range from early steps in vector production to evaluation of insertion sites post-transduction. We have evaluated an FDA approved DNase for removal of residual plasmid DNA during vector production, developed novel non-integrating lentiviral vectors and employed modified insertion site analysis post-transduction to improve the safety of lentiviral vector mediated gene therapy. To prevent the exposure of gene therapy patients to HIV-1 DNA it is essential to remove residual plasmid DNA during vector production. We evaluated a recombinant human DNase which has been FDA approved for use in patients as an alternative to a bacterially derived DNase. Our results indicate this DNase is an effective alternative with a potentially safer profile for use in patients. The ability of lentiviral vectors to stably integrate their genome into a host cell’s DNA can have negative side-effects due to the risk of insertional mutagenesis. Non-integrating lentiviral vectors have been developed to alleviate this risk in applications where integration is not necessary. However, a low frequency of illegitimate integration persists when using these vectors. We have developed a novel non-integrating vector mutation and evaluated the efficacy of combining it with other mutations for reducing the frequency of illegitimate integration. We demonstrate that combining mutations that inhibit integration can further reduce the frequency of illegitimate integration. Several methodologies have been developed for evaluating the insertion sites of normal integrating lentiviral vectors. Illegitimate integration by non-integrating vectors demonstrates mechanisms which result in insertions and/or deletions at the vector-genome junction. Current methods lack the sensitivity to account for these variables in a high-throughput manner. We have adapted modifications to current methods to improve the capture of these variable insertion sites for analysis. The results of these studies improve the safety of lentiviral vector mediated gene therapy by improving the purity of the vector product, providing a safer vector for non-integrase mediated applications, and allowing more sensitive analysis of insertion sites post-transduction.Item APOE4 Drives Impairment in Astrocyte-Neuron Coupling in Alzheimer's Disease and Works Through Mechanisms in Early Disease to Influence Pathology(2023-05) Brink, Danika Marie Tumbleson; Lamb, Bruce; Bissel, Stephanie; Herbert, Brittney-Shea; Landreth, Gary; Puntambekar, Shweta; Saykin, Andrew; Zhang, ChiAlzheimer’s disease (AD) is a neurodegenerative disorder resulting in progressive memory loss, brain atrophy, and eventual death. AD pathology is characterized by the accumulation of neurotoxic amyloid-beta (Aβ) plaques, synapse loss, neurofibrillary tangles (NFTs), and neurodegeneration. The APOE4 allele is associated with a 3-fold increased risk for AD and results in increased Aβ plaque deposition, reduced Aβ clearance, and reduced synaptic plasticity. Although APOE expression is upregulated in microglia in AD, APOE is expressed primarily by astrocytes in the CNS. It is not well understood how astrocytic APOE drives the mechanisms that result in worsened AD outcomes. Here, digital spatial profiling and bioinformatics data suggest that APOE4 causes transcriptional dysregulation in early AD and may disrupt neuronal processes via astrocytes. Whole transcriptome data from plaque and non-plaque regions in the cortices and hippocampus of 4- and 8-month-old AD model mice expressing humanized APOE4/4 or APOE3/3 (control) were analyzed. Transcriptional dysregulation was increased in APOE4/4 AD mice compared to that in APOE3/3 at 4 but not 8 months of age, suggesting that early dysregulation of APOE4-driven disease mechanisms may shape degenerative outcomes in late-stage AD. Additionally, APOE4/4 potentially functions via plaque-independent mechanisms to influence neuronal function in early AD before the onset of pathology. Single-nuclei RNA sequencing data were obtained from human post-mortem astrocytes and the bioinformatic analyses revealed a novel astrocyte subtype that highly expresses several top genes involved in functional alterations associated with APOE4, including neuronal generation, development, and differentiation, and synaptic transmission and organization. Overall, our findings indicate that APOE4 may drive degenerative outcomes through the presented astrocyte candidate pathways. These pathways represent potential targets for investigations into early intervention strategies for APOE4/4 patients.Item Cognitive dysfunction in cancer: Neuroimaging and genetic approaches to identify biological mechanisms(2015-04-22) Nudelman, Kelly N. H.; Saykin, Andrew J.; Foroud, Tatiana M.; McDonald, Brenna Cathleen; Schneider, Bryan Paul; Shen, LiAlthough cancer and treatment-associated cognitive dysfunction has been well-documented in the literature, much work remains to elucidate the biological mechanisms driving this effect, hampering current therapeutic efforts. To address this gap, we first reviewed studies utilizing neuroimaging to characterize cognitive dysfunction in cancer, as studies of neurodegenerative diseases point to neuroimaging as a sensitive measure of cognitive dysfunction. This review highlighted the need for longitudinal imaging studies of cancer and treatment-related changes in cerebral structure and function. Subsequently, we utilized multimodal neuroimaging techniques in a female breast cancer cohort to investigate the longitudinal impact of cancer and chemotherapy treatment on cerebral perfusion and gray matter. Our findings indicate that chemotherapy is associated with elevated perfusion, primarily in posterior brain regions, as well as depressed frontal perfusion associated with decreased frontal gray matter density. This pattern of results suggests the involvement of multiple mechanisms of chemotherapy-induced cognitive dysfunction. We also investigated the relationship of cognitive dysfunction and chemotherapy-induced peripheral neuropathy (CIPN), another type of chemotherapy-related nervous system sequelae, again utilizing multimodal, longitudinal neuroimaging, and found that peripheral neuropathy symptoms following chemotherapy were associated with changes in cerebral perfusion and gray matter density. Together, these findings support the hypothesis that multiple biological mechanisms drive cancer and treatment-related cognitive dysfunction. Interestingly, although cancer is associated with cognitive dysfunction, epidemiological studies have shown that cancer and Alzheimer's disease (AD) are inversely correlated. To extend our imaging analysis beyond breast cancer, we leveraged the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort to investigate the inverse relationship of cancer and AD and investigate the impact of both of these diseases on gray matter density. We found that though the inverse relationship of these diseases was replicated in the ADNI cohort, cancer history was associated with lower gray matter density, similar to findings from breast cancer studies, independent of AD diagnostic group. Finally, we reviewed microRNA studies, as microRNAs are important regulators of many cell signaling pathways and have been actively investigated in relation to both diseases. This review suggests several pathways that may be driving the inverse association and may contribute to cognitive dysfunction.Item Computational modeling for identification of low-frequency single nucleotide variants(2015-11-16) Hao, Yangyang; Liu, Yunlong; Edenberg, Howard J.; Li, Lang; Nakshatr, HarikrishnaReliable detection of low-frequency single nucleotide variants (SNVs) carries great significance in many applications. In cancer genetics, the frequencies of somatic variants from tumor biopsies tend to be low due to contamination with normal tissue and tumor heterogeneity. Circulating tumor DNA monitoring also faces the challenge of detecting low-frequency variants due to the small percentage of tumor DNA in blood. Moreover, in population genetics, although pooled sequencing is cost-effective compared with individual sequencing, pooling dilutes the signals of variants from any individual. Detection of low frequency variants is difficult and can be cofounded by multiple sources of errors, especially next-generation sequencing artifacts. Existing methods are limited in sensitivity and mainly focus on frequencies around 5%; most fail to consider differential, context-specific sequencing artifacts. To face this challenge, we developed a computational and experimental framework, RareVar, to reliably identify low-frequency SNVs from high-throughput sequencing data. For optimized performance, RareVar utilized a supervised learning framework to model artifacts originated from different components of a specific sequencing pipeline. This is enabled by a customized, comprehensive benchmark data enriched with known low-frequency SNVs from the sequencing pipeline of interest. Genomic-context-specific sequencing error model was trained on the benchmark data to characterize the systematic sequencing artifacts, to derive the position-specific detection limit for sensitive low-frequency SNV detection. Further, a machine-learning algorithm utilized sequencing quality features to refine SNV candidates for higher specificity. RareVar outperformed existing approaches, especially at 0.5% to 5% frequency. We further explored the influence of statistical modeling on position specific error modeling and showed zero-inflated negative binomial as the best-performed statistical distribution. When replicating analyses on an Illumina MiSeq benchmark dataset, our method seamlessly adapted to technologies with different biochemistries. RareVar enables sensitive detection of low-frequency SNVs across different sequencing platforms and will facilitate research and clinical applications such as pooled sequencing, cancer early detection, prognostic assessment, metastatic monitoring, and relapses or acquired resistance identification.Item Consequences of telomerase inhibition and telomere dysfunction in BRCA1 mutant cancer cells(2014-03-12) Phipps, Elizabeth Ann; Herbert, Brittney-Shea; Grimes, Brenda R.; Sledge, George W.; Turchi, John; White, Kenneth E.Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.Item DECODING THE TRANSCRIPTIONAL LANDSCAPE OF TRIPLE-NEGATIVE BREAST CANCER USING NEXT GENERATION WHOLE TRANSCRIPTOME SEQUENCING(2012-03-16) Radovich, Milan; Schneider, Bryan P.; Flockhart, David A.; Ivan, Mircea; Herbert, Brittney-Shea; Grimes, Brenda R.; Nakshatri, HarikrishnaTriple-negative breast cancers (TNBCs) are negative for the expression of estrogen (ER), progesterone (PR), and HER-2 receptors. TNBC accounts for 15% of all breast cancers and results in disproportionally higher mortality compared to ER & HER2-positive tumours. Moreover, there is a paucity of therapies for this subtype of breast cancer resulting primarily from an inadequate understanding of the transcriptional differences that differentiate TNBC from normal breast. To this end, we embarked on a comprehensive examination of the transcriptomes of TNBCs and normal breast tissues using next-generation whole transcriptome sequencing (RNA-Seq). By comparing RNA-seq data from these tissues, we report the presence of differentially expressed coding and non-coding genes, novel transcribed regions, and mutations not previously reported in breast cancer. From these data we have identified two major themes. First, BRCA1 mutations are well known to be associated with development of TNBC. From these data we have identified many genes that work in concert with BRCA1 that are dysregulated suggesting a role of BRCA1 associated genes with sporadic TNBC. In addition, we observe a mutational profile in genes also associated with BRCA1 and DNA repair that lend more evidence to its role. Second, we demonstrate that using microdissected normal epithelium maybe an optimal comparator when searching for novel therapeutic targets for TNBC. Previous studies have used other controls such as reduction mammoplasties, adjacent normal tissue, or other breast cancer subtypes, which may be sub-optimal and have lead to identifying ineffective therapeutic targets. Our data suggests that the comparison of microdissected ductal epithelium to TNBC can identify potential therapeutic targets that may lead to be better clinical efficacy. In summation, with these data, we provide a detailed transcriptional landscape of TNBC and normal breast that we believe will lead to a better understanding of this complex disease.Item Development of Cancer-Genomics-Guided Precision Immunotherapy for Triple-Negative Breast Cancer(2023-05) Sun, Yifan; Lu, Xiongbin; Kaplan, Mark H.; Hopewell, Emily L.; Zhang, Chi; Yang, KaiTriple-negative breast cancer (TNBC), which accounts for 15-20% of all breast cancers, is highly aggressive and metastatic with the poorest overall rates. While surgery, radiation, and chemotherapy remain the main treatment options, TNBC represents an unmet medical need for better treatment strategies. Tremendous efforts have been made to develop effective therapies over the past years. However, TNBC treatment options are still very limited due to the lack of good drug targets and the low response rate of current therapies. In this study, we developed two different strategies to treat TNBC based on its cancer genomic features: 1) heterozygous loss of chromosome 17p (17p loss) and 2) high mutation load. 17p loss is one of the most frequent genomic events in breast cancer including TNBC, rendering cancer cells vulnerable to the inhibition of POLR2A via α-amanitin (POLR2A-specific inhibitor). Here, we developed a new drug T-Ama (α-amanitin-conjugated trastuzumab) targeting HER2-low TNBC with 17p loss by combining the effects of α-amanitin and trastuzumab (HER2+ breast cancer therapy). Our results showed that T-Ama exhibited superior efficacy in treating HER2-low TNBC with 17p loss in vitro and in vivo, and surprisingly induced immunogenic cell death (ICD) which further enhanced T cell infiltration and cytotoxicity levels and delivered greater efficacy in combination with immune checkpoint blockade therapy. Collectively, the therapeutic window created by 17p loss and HER2 expression will make HER2-low TNBC clinically feasible targets of T-Ama. As another genetic feature of TNBC, the higher genomic instability and mutational burden results in more neoantigens presented on MHC-I, along with the higher level of tumor-infiltrating T cells, making TNBC a perfect model for immunotherapy compared to the other breast cancer subtypes. Here, we designed a deconvolution-algorithm-derived library screening to find new therapeutic targets and identified PIK3C2α as a key player that determines MHC-I turnover and reduces the MHC-I-restricted antigen presentation on tumor cells. In preclinical models, inhibition of PIK3C2α profoundly suppressed breast tumor growth, increased tumor-infiltrating CD8+ T cells, and showed high potential enhancing the efficacy of anti-PD-1 therapy, suggesting that PIK3C2α is a potential therapeutic target for TNBC immunotherapy.Item DISHEVELLED-ASSOCIATED ACTIVATOR OF MORPHOGENESIS 1 (DAAM1) IS REQUIRED FOR HEART MORPHOGENESIS(2010-02-02T19:55:35Z) Li, Deqiang; Shou, Weinian; Field, Loren J.; Payne, R. Mark; Zhang, XinDishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, has been implicated in the non-canonical Wnt mediated Planar Cell Polarity (PCP) signaling pathway. Although the studies in Drosophila Daam1 and Xenopus Daam1 generated inconsistent conclusions regarding the function of Daam1, the biological function of mammalian Daam1 was not evaluated. In this study, we used a mouse promoter trap technology to create Daam1 deficient mice to analyze the role of Daam1 in embryonic development and organogenesis. Daam1 is highly expressed in the developing heart. The majority of Daam1 mutant mice died between embryonic day 14.5 and birth, exhibiting a variety of heart defects, which include ventricular noncompaction, ventricular septal defects, and double outlet right ventricle. About 10% mutant mice survive to adulthood, and these survivors do not show significantly compromised heart function based on echocardiographic analyses. However, all of these mutant survivors have ventricular noncompaction with a range of severities. A conditional rescue experiment using a cardiac specific Cre mouse line, Nkx2-5Cre, confirmed that the cardiac defects are the primary cause of death in Daam1 mutants. Both in vivo and ex vivo analyses revealed that Daam1 is essential for regulating non-sarcomeric filamentous actin assembly in cardiomyocytes, which likely contributes to cardiac morphogenesis and ventricular wall maturation. Biochemical studies further suggested that Daam1 is not a key signaling component in regulating the activation of small GTPases, such as RhoA, Rac1 and Cdc42. In conclusion, our studies demonstrated that Daam1 is essential for cardiac morphogenesis likely through its regulation of cytoskeletal architecture in the developing cardiomyocytes.Item Familial Studies in Whole Exome and Genome Sequencing(2015) Farlow, Janice L.; Foroud, TatianaPopulation genetics has been revolutionized by the advent of high-throughput sequencing (HTS) methods in the 21st century. Modern day sequencers are now capable of sequencing entire exomes and genomes at unprecedented speed and accuracy. An explosion of bioinformatics software and data analysis tools now makes sequencing accessible for gene discovery in both rare Mendelian and complex disease. Family-based sequencing studies in particular have great potential for elucidating the genetic basis for many more diseases. We apply both whole exome and genome sequencing to three different cases of familial disease: intracranial aneurysm (IA), Parkinson disease (PD), and X-linked ataxia dementia (XLAD). IA and PD are both common, complex traits that inflict a devastating disease burden worldwide, mostly due to few effective therapeutic interventions. Little of the heritability of both IA and PD has been explained to date, especially as it relates to the impact of rare variation on disease. XLAD is an extremely rare neurological disease described thus far in one kindred. Although promising results have been achieved through previous genetic study designs, the causative gene has not yet been identified. For all three diseases, HTS offers an opportunity to explore the role of rare variation in disease pathogenesis. In each study, we explore the opportunities and challenges of family-based HTS for different disease models. The work presented herein contributes effective practices for study design, analysis, and interpretation in a rapidly growing field still replete with questions about how best to implement HTS in studying familial disease.