Date of Award

6-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Tanja Gruber, PhD

Committee

Ronald N. Laribee, PhD Stacey K. Ogden, PhD Paul G. Thomas, PhD Gerard P. Zambetti, PhD

Keywords

Immunotherapy, Leukemia, Transcription Factor

Abstract

CBFA2T3-GLIS2 is the most prevalent fusion oncogene in pediatric acute megakaryoblastic leukemia in patients without Down syndrome (non-DS-AMKL) and is associated with an event free survival of only 8% even with high intensity chemotherapy and stem cell transplant in first remission. A cryptic inversion event on chromosome 16 joins the three nervy homology regions (NHR) of CBFA2T3 to the five zinc fingers of GLIS2. This configuration enables the encoded chimeric transcription factor to bind GLIS consensus sequences throughout the genome and recruit transcriptional activators and repressors to alter gene expression and enhance self-renewal capability. Few cooperating mutations have been identified in patients harboring this fusion which suggests it is the sole oncogenic driver. The molecular mechanism by which CBFA2T3-GLIS2 drives leukemogenesis is not well understood. Further, there are currently no studies exploring the immunogenicity of the fusion positive AMKL cells and immunotherapeutic approaches are lacking. To address these knowledge gaps, I developed two mouse models to study transcriptional regulation by the CBFA2T3-GLIS2 fusion oncogene and the ability of the immune system to recognize this malignancy with a goal of identifying therapeutic vulnerabilities for future translational studies. To understand transcriptional regulation by the fusion, I first turned to the literature. Studies on the wild type CBFA2T3 and GLIS2 proteins have demonstrated interactions with the transcriptional regulators ETO and CtBP1, respectively. Further, p300 has been shown to play a role in transcriptional regulation imparted by both transcription factors. I therefore hypothesized the fusion promotes transcriptional activation when the histone acetyltransferase p300 and the transcription factor ETO are recruited through NHR1 and NHR2, respectively. When the co-repressor CtBP1 is recruited through the PXDLS motif, located in the GLIS2 portion of the fusion, transcriptional repression predominates. Association of these co-factors with the fusion was confirmed through co-immunoprecipitations. Site-directed mutagenesis was then used to systematically delete NHR1 and NHR2 and mutate the PXDLS motif to evaluate the resultant effects on leukemogenesis imparted by the fusion. Murine models harboring the CBFA2T3-GLIS2 fusion without cooperating mutations have been unsuccessful and patient-derived xenograft (PDX) models are limited and difficult to manipulate. Therefore, I developed a novel humanized model of CBFA2T3-GLIS2 driven leukemia to understand the functional consequence of the mutant constructs. CD34+ stem cells were isolated from human cord blood and transduced with a lentivirus construct encoding the fusion and a GFP reporter. The cells were then differentiated to megakaryoblasts using human TPO and IL1β and sorted for purity prior to transplantation into immunodeficient NSG-SGM3 recipient mice. The fusion positive human primary megakaryoblasts induced a serially transplantable leukemia within 180 days that recapitulates CBFA2T3-GLIS2 positive patient samples on a transcriptional and translational level. Using the model, I found the fusion co-occupies sites throughout the genome with ETO and CtBP1 and induces upregulation of oncogenic signaling pathways including Hedgehog, JAK/STAT, Notch, and RUNX. Loss of NHR2 disrupted ETO association with the fusion, decreased fusion homodimerization, and abrogated leukemogenesis in vivo. I observed downregulation of JAK/STAT, Notch, and Hedgehog signaling pathways in cells with a fusion construct that lacks NHR2, suggesting these pathways may play a critical role in transformation. Future studies using CRISPR editing technology will be used to further elucidate the role of these pathways in AMKL transformation. An alternative to identifying proteins and biologic pathways necessary for transformation to target therapeutically is the use of immunotherapeutic approaches. In addition to a lack of knowledge about the CBFA2T3-GLIS2 transcriptional complex, there are currently no studies exploring the immunogenicity of the fusion positive AMKL cells. Since chemotherapy and stem cell transplantation fail to cure this disease, innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven’t been realized in acute myeloid leukemia (AML) due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which uses heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by HLA molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, I first interrogated potential neoantigens spanning the fusion junction using the algorithm from NetMHCcons which identified two significant peptides. I then utilized immunopeptidomics to identify HLA class I presented peptides on the surface of AMKL PDX cells. One of the top hits was a peptide that corresponds to the 5’ untranslated region (UTR) of bone morphogenetic protein 2 (BMP2). Although its role in leukemia is not well described, BMP2 is aberrantly upregulated in CBFA2T3-GLIS2 positive AMKL and required for serial replating in colony forming unit assays; therefore, this peptide is of great interest as a candidate leukemia-associated antigen. To identify potential AMKL directed effector CD8+ T cells, I developed a novel HLA class I exact matched PBMC-humanized CBFA2T3-GLIS2 positive AMKL PDX murine model. I observed a variable response in leukemia burden reduction, with the greatest responses correlating with an increase in granzyme A production and a decrease in TCR diversity; consistent with a potential clonal expansion of leukemia-specific effector CD8+ T cells. Single TCR expressing stable cell lines will be generated for the top expanded clones and used to assess leukemia clearance in the model and to measure specificity and functionality for the putative candidate antigens through tetramer staining and peptide stimulation assays, respectively. These novel studies have shed light on the immunogenicity of CBFA2T3-GLIS2 mediated AMKL and have the potential to contribute to the development of a TCR-T immunotherapy in the setting of minimal residual disease to provide an alternative, effective treatment for this chemotherapy resistant malignancy.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-7292-4347

DOI

10.21007/etd.cghs.2022.0597

2022-013-Garfinkle-DOA.pdf (209 kB)
Declaration of Authorship

Supplemental Immunopeptidomics Data for Chapter 3.xlsx (26 kB)
Supplemental Immunopeptidomics Data for Chapter 3

Supplemental Mouse Data for Chapter 2.xlsx (21 kB)
Supplemental Mouse Data for Chapter 2

Supplemental Mouse Data for Chapter 3.xlsx (14 kB)
Supplemental Mouse Data for Chapter 3

Supplemental Sequencing Data for Chapter 2.xlsx (3935 kB)
Supplemental Sequencing Data for Chapter 2

Available for download on Friday, July 14, 2023

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