Abstract Changes in gene expression and subsequent editing patterns of APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like) cytosine deaminases have been shown to be present in ...solid tumor cancer evolution, however, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation, especially in the hematopoietic niche. Building on advanced sequencing data of healthy, pre-leukemic, and myeloproliferative neoplasm patient samples, we seek to uncover normal and malignant patterns of APOBEC3C overexpression in hematopoietic cells. Overexpression of lentiviral APOBEC3C and an editase-mutated APOBEC3C in normal cord blood, healthy bone marrow, and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of deaminase dysregulation in hematopoietic cells. In addition to APOBEC3, we are exploring the upregulation of adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown that these two innate immune deaminases are synchronously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. These overexpression studies show novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis, which can be cross-referenced to gene expression and mutation signatures seen in hematopoietic malignancies and solid tumor cancers. In addition to mutation patterns, the regulation of LINE-1 elements has been shown to be another important function of APOBEC3 enzymes. In these studies, we examine the effects of APOBEC3 overexpression on repetitive element expression in hematopoietic cell populations and have found that there are strong cell type and context patterns across the APOBEC3 family and between cell types within the hematopoietic system. We will use this comprehensive study of APOBEC3 deregulation to uncover predictive biomarkers of malignant transformation. Citation Format: Jane Marie Isquith, Thomas Whisenant, Jessica Pham, Ludmil Alexandrov, Catriona Jamieson. Hallmarks of APOBEC3 mutagenesis in normal, pre-leukemic, and myeloproliferative neoplasm cell populations abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1485.
Abstract
Dysregulation of inflammatory cytokine responsive APOBEC3 cytosine deaminases has been shown to be a contributing factor in cancer evolution, presenting as gene expression changes and ...inclusion of distinct C-to-T mutation patterns. However, the context specificity and mechanisms by which APOBEC3 enzymes promote cancer initiation and progression require further elucidation. Lentiviral overexpression of APOBEC3C and an editase deficient APOBEC3C mutant in healthy cord blood, bone marrow and MPN patient hematopoietic stem/progenitor cells (HSPCs) allows us to study the effects of innate immune deaminase dysregulation in the hematopoietic niche. We are focusing on the upregulation of APOBEC3C and adenosine deaminase acting on RNA1 (ADAR1), as we have previously shown them to be contemporaneously upregulated in the high-risk myelofibrosis (MF) stem cell population compared to normal aged bone marrow. We can compare these novel differential gene expression changes, RNA hyper-editing sites, and DNA mutation signatures induced by APOBEC3 mutagenesis to abnormalities seen in both hematopoietic malignancies and solid tumor cancers. Gene set enrichment analysis (GSEA) performed on this dataset has exposed numerous deregulated pathways brought on by exaggerated levels of APOBEC3, including changes in splicing pathways. To further investigate the complex relationship between splicing and deaminase deregulation, we treated myeloproliferative neoplasm patient samples and normal HPSCs with Rebecsinib (also known as 17S-FD-895), a pharmacologically stable, potent, and selective small molecule splicing modulator, which interestingly caused significant downregulation of APOBEC3C. We will continue to investigate these findings as a potential target to correct the dysregulation seen in MPN progression. Consequently, we aim to use these findings to identify predictive biomarkers and druggable targets of leukemic initiation progression.
Citation Format: Jane Marie Isquith, Jessica Pham, Thomas Whisenant, Larisa Balaian, Catriona Jamieson. Molecular mechanisms of RNA and DNA editing in leukemic transformation of hemopoietic stem and progenitor 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 123.
Abstract
Inflammatory cytokine responsive APOBEC3 cytidine deaminases have been studied extensively with regard to innate immunity and more recently during cancer evolution. However, the mechanisms ...by which the APOBEC3 enzymes promote cancer initiation and progression in the malignant microenvironment remains to be investigated, especially in hematopoietic malignancies. Through whole genome and whole transcriptome sequencing analyses of MPN patient samples FACS sorted into stem and progenitor populations, we have found a cell type and context specific nature of these enzymes, notably the upregulation of APOBEC3C (A3C) in the high-risk Myelofibrosis (MF) stem cell population as compared to normal aged counterparts. Through lentiviral overexpression of each APOBEC3 enzyme, we can now study the effects of changes in APOBEC3 transcript level in relation to the known changes in expression seen in many cancers, focusing on the upregulation of A3C. Using these techniques, we have identified novel RNA and DNA editing targets, as well as differential gene expression patterns of each APOBEC3 in normal CD34+ cord blood and aged normal bone marrow. Gene set enrichment analysis (GSEA) performed on this dataset has exposed numerous deregulated pathways brought on by exaggerated levels of APOBEC3, including changes in splicing pathways. In addition, novel identification of the relationship between A3C and ADAR1, another innate immune deaminase, has important implications in initiation and prognosis of MPNs. Both A3C and ADAR1 transcript levels are elevated in high risk MF stem cells, and co-immunoprecipitation studies reveal a direct binding of the enzymes. Furthermore, we are able to study the detailed effects of editing by both A3C and ADAR1 using editase-deficient mutant constructs, allowing for a mechanistic look into the role of these deaminases and their deregulation in vitro and in vivo using patient samples and humanized mouse models. This novel connection, as well as the role of A3C in initiation and progression of hematopoietic malignancies will continued to be studied using this system to elucidate effects on proliferation, differentiation, self-renewal, and changes to the cell cycle, in hopes of creating both a marker of early detection and potentially a druggable target.
Citation Format: Jane Marie Isquith, Adam Mark, Jessica Pham, Mary Donohoe, Luisa Ladel, Catriona Jamieson. Effects of innate immune deaminase deregulation on initiation and progression of myeloproliferative neoplasms abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 899.
Mesenchymal stem cells (MSC) have been isolated from equine tissues, most notably adipose and bone marrow. On the basis of ability for self‐renewal, molecular marker expression and differentiation ...potential, we characterized MSCs isolated from a tissue not yet explored in the equine. After isolation, MSCs were maintained in culture for 25 passages before senescence was observed. Reverse transcription‐PCR revealed embryonic transcription factor Oct4 mRNA, as well as mRNA for fucosyltransferase 4 and ST3 B‐galactoside α 2,3 sialyltransferase 2, two enzymes responsible for synthesis of embryonic surface markers SSEA1 and SSEA4, respectively. Treatment with insulin, dexamethasone, indomethacin and isobutylmethylxanthine led to increased oil red O staining (P<0.05) indicating successful adipogenic differentiation. These data indicate the stem‐like nature of these cells and their successful isolation from this tissue source. However, further characterization is needed to determine their full potential.
Partially funded by College of Agriculture, Food and Environmental Science, Cal Poly San Luis Obispo
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The deregulation of the cell cycle is important in the leukemic transformation and in the continued discovery of more efficient targeted therapies. After elucidation of the role of the RNA editor ...ADAR1 in the malignant reprogramming of Chronic Myeloid Leukemia (CML) progenitors to leukemia stem cells (LSCs) during the chronic phase to blast crisis stages of the disease, current research seeks to uncover the mechanism of action of ADAR1 in this process. To study the potential role ADAR1 plays in the changes to cell cycle during this disease progression, a novel Fucci (fluorescent ubiquitination-based cell cycle indicator) system was introduced into the CML cell line K562 through stable transduction of both Fucci vectors. This allows for the visualization of the cell cycle via FACS (fluorescence activated cell sorting), and fluorescent live imaging. This system was validated for use in therapeutic CML research through preliminary evaluation of ADAR1 activity in specific cell cycle stages as well as the effects of a potentially novel CML therapy, 8-azaadenosine, on the cell cycle. Completion of this work shows successful creation of a novel generation of Fucci cell line; which will aid in the clarification of the changes which occur in the cell cycle during CML disease progression, as well as the effects of CML targeted therapies on cell cycle regulation though quantitative and qualitative analysis.
The deregulation of the cell cycle is important in the leukemic transformation and in the continued discovery of more efficient targeted therapies. After elucidation of the role of the RNA editor ...ADAR1 in the malignant reprogramming of Chronic Myeloid Leukemia (CML) progenitors to leukemia stem cells (LSCs) during the chronic phase to blast crisis stages of the disease, current research seeks to uncover the mechanism of action of ADAR1 in this process. To study the potential role ADAR1 plays in the changes to cell cycle during this disease progression, a novel Fucci (fluorescent ubiquitination-based cell cycle indicator) system was introduced into the CML cell line K562 through stable transduction of both Fucci vectors. This allows for the visualization of the cell cycle via FACS (fluorescence activated cell sorting), and fluorescent live imaging. This system was validated for use in therapeutic CML research through preliminary evaluation of ADAR1 activity in specific cell cycle stages as well as the effects of a potentially novel CML therapy, 8-azaadenosine, on the cell cycle. Completion of this work shows successful creation of a novel generation of Fucci cell line; which will aid in the clarification of the changes which occur in the cell cycle during CML disease progression, as well as the effects of CML targeted therapies on cell cycle regulation though quantitative and qualitative analysis.
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