Kristen Balogh, PhD, 2018

The role of MIF in anti-tumor immune responses and metastatic niche formation in breast cancer

Abstract:

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Kiley Knapp, PhD, 2018

Evaluation of SAS1B as an Immunotherapeutic Target for the Treatment of Cancer

Abstract:

Immunotherapeutic options for the treatment of cancer, which remains a major global health challenge, offer the allure of greater tumor specificity and less associated toxicity than is typically achieved with traditional chemo- and radio-therapeutic strategies. The success of some immunotherapies, such as ADCs and CAR-Ts, rely on identification and selection of targets which are highly tumor-specific with limited or no expression in normal tissues. Cancer germline antigens represent potential ideal targets for targeted immunotherapy as CGAs are expressed in cancer cells but show limited or no expression among normal tissues. Although many cancer-testis antigens have been described, SAS1B is the first, and only, cancer-oocyte antigen identified to-date. Owing to the limited expression of SAS1B among normal tissue combined with expression in a number of cancer indications, we propose that SAS1B is an attractive immunotherapeutic target. We have shown that, in addition to previous work published in female reproductive cancers, SAS1B is expressed in a majority of pancreatic and head and neck cancers. SAS1B localized to both the cytoplasm and the cell surface in PDAC and HNSCC cell lines by IIF and flow cytometry, suggesting potential utility of SAS1B targeted immunotherapeutic strategies. Furthermore, an ADC targeting SAS1B administered to pancreatic cancer cell lines was internalized and subsequently caused significant cell death in a manner correlated with SAS1B cell surface expression. Thus, SAS1B represents a novel therapeutic target for the treatment of PDAC and HNSCC. These data support further development of a SAS1B-ADC including in vivo assessment using mouse xenograft systems. Although we have shown proof of concept that SAS1B-ADC induces cytotoxicity in pancreatic cancer cell lines, addressing multiple fundamental biological questions which remain regarding SAS1B expression will also inform production of SAS1B targeted therapies. For example, we have identified six ASTL splice variants in cancer, known as SV-A to SV-F, and have shown differential cellular localization of recombinant SV-A and SV-C proteins (cell surface vs. cytoplasm, respectively). Further studies characterizing major SAS1B protein isoform(s) expressed at the cell surface in cancers may lead to development of more effective immunotherapies utilizing mAbs generated against cell surface, cancer-associated form(s) of SAS1B. Our work suggests SAS1B expression in a broad range of cancer indications and demonstrates efficacy of a SAS1B-ADC in vitro, thus supporting further assessment of SAS1B as an immunotherapeutic target for the treatment of cancer.

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Ryan Haskins, PhD, 2018

Klf4 Has an Unexpected Protective Role in Perivascular Cells Within the Microvasculature

Abstract:

Recent smooth muscle cell (SMC) lineage tracing studies have revealed that SMCs undergo remarkable changes in phenotype during the development of atherosclerosis. Of major interest, this work demonstrated that Klf4 in SMCs is detrimental for overall lesion pathogenesis in that SMC-specific conditional knockout of Klf4 resulted in smaller, more stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage-like and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. Herein the hypothesis that Klf4 dependent SMC transitions play an important role in tissue repair following injury was tested. Utilizing SMC-specific lineage tracing mice +/- simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart following ischemia-reperfusion injury (IRI) express KLF4 and transition to a Klf4 dependent macrophage-like state and a Klf4 independent myofibroblast-like state. Moreover, SMC-Klf4 knockout mice had exacerbated heart failure following IRI. Surprisingly, significant cardiac dilation was observed in SMC-Klf4 knockout mice prior to IRI. This cardiac dilation was accompanied by a reduction in peripheral resistance, as evidenced by a reduction in blood pressure, an increase in blood flow, and a larger passive diameter of mesenteric resistance arteries as measured by pressure myography. KLF4 ChIP-Seq analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways previously shown to be important for perivascular cell investment including PDGF and FGF. Moreover, microvascular tissue beds in SMC-Klf4 knockout mice had gaps in lineage traced SMC coverage along the resistance arteries and exhibited increased permeability. Taken together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs, including being required for normal SMC function and coverage of resistance arteries.

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Kasey Jividen, PhD, 2018

The Interface Between Androgen Signaling and DNA Repair in Prostate Cancer

Abstract:

Prostate cancer remains the second leading cause of cancer-related deaths in American men. The cellular processes of the prostate, including prostate cancers, are governed by a nuclear hormone receptor known as the androgen receptor (AR). Androgen binding to AR controls the expression of thousands of target genes in prostate cells. These genes influence many important signaling pathways including cell growth, survival, metabolism, and DNA repair. Early disease interventions that include androgen deprivation therapies (ADTs) are successful; however, these therapies eventually become ineffective, and to this day, castrate resistant prostate cancer (CRPC) treatment remains an unmet clinical need. CRPC is marked by changes in signaling pathways such as cell growth, motility, and DNA repair. Understanding these changes and determining crucial regulators of these changes will provide alternative and improved therapies for CRPC. Because recent evidence has shown that androgen and DNA repair signaling are closely linked processes, we conducted a comprehensive assessment of expression level changes of DNA repair factors by androgen signaling in multiple prostate cancer cell lines, in disease progression models of prostate cancer, and using prostate cancer patient data. Using high throughput sequencing, we uncovered a previously unreported DNA repair gene set that was regulated by androgen signaling in multiple prostate cancer cell lines. Genomic analyses of these prostate cancer cell lines also provided key information related to the mutation status of genes encoding DNA repair proteins. This interrogation of prostate cancer preclinical models is likely to inform future studies in the field. DNA repair factors are not only found to be regulated by androgen-mediated transcription, but other laboratories have shown that DNA repair machinery is also required for transcriptional activities; this includes the activities of nuclear hormone receptors. As part of this study, we tested a new inhibitor that targets one such DNA repair factor. We used mirin, a small molecule inhibitor against the DNA-damage sensing protein MRE11. We found that mirin treatment of prostate cancer cells significantly inhibits the transcription of several androgen-regulated genes. Treatment with mirin also resulted in cell growth inhibition and cell cycle arrest of prostate cells. In hormone-driven cancers like breast and prostate, clinical trials recently showed that the molecular targeting of the DNA repair protein Parp1 with Olaparib improved patient outcomes in individuals with DNA repair gene mutations. Other Parp family members have emerged as central players in disease development processes like chemo-resistance and cell survival. Parp9 is one family member that along with the heterodimer partner, Dtx3L, function in the DNA damage response. In this study, we characterized the dual enzymatic nature of this Dtx3L/Parp9 heterodimer in the context of prostate cancer. Here we report the previously unknown ADP-ribosylation activity of Parp9 towards the C-terminal end of ubiquitin as well as the regulation of the E3 ubiquitin ligase activity of Dtx3L by NAD+. These activities were shown to be related to the DNA damage response, particularly the NHEJ repair pathway. The role of Dtx3L and Parp9 in prostate cells also includes a direct interaction with AR via the macrodomains of Parp9. Dtx3L/Parp9 association with AR depends on AR ADP-ribosylation by Parp7. Our data also showed that Parp7 is another apparent target of the clinically available Parp inhibitor, Olaparib. Overall, the experiments from this study show novel relationships between DNA repair signaling and androgen signaling, and provide preclinical results that will support and potentially complement current therapies for prostate cancer.

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Paige Kulling, PhD, 2018

Characterizing and Targeting the Interferon-Gamma Signaling Pathway in T Cell Large Granular Lymphocyte Leukemia

Abstract:

T cell large granular lymphocyte leukemia (T-LGLL) is a rare, chronic hematological malignancy characterized by clonal proliferation of cytotoxic CD8+ T cells due to defective apoptosis. Currently there is no cure and the main treatment options are broad immunosuppressant therapies for management of symptoms. The vast majority of T-LGLL patients require treatment at some point and thus there are efforts to identify and evaluate novel and specific therapeutics for T-LGLL. One such area of investigation is the regulation of interferon-gamma signaling. IFN-gamma is an inflammatory cytokine that is associated with worse disease progression and symptomology in multiple cancers and autoimmune diseases. Excess IFN-gamma, in the absence of infection, inhibits proliferation and induces apoptosis of healthy peripheral blood mononuclear cells (PBMCs), contributing to disease state. As a result of this, it is crucial to reduce IFN-gamma production in cancers, including T-LGLL, where patients have significantly elevated circulating levels of this pro-inflammatory cytokine compared to healthy donors. Calcitriol, the active form of vitamin D, has shown promise as an inhibitor of IFN-gamma production. Thus, we first turned our attention to evaluating calcitriol for use as an IFN-gamma inhibitor in T-LGLL. We initially assessed the effects of 24 h calcitriol treatment on TL-1 cells, the patient-derived cell line model of T-LGLL (Chapter 3). We found that calcitriol significantly decreased IFN-gamma secretion and activation of signal transducer and activator of transcription 1 (STAT1), a transcription factor that becomes activated via phosphorylation of tyrosine residue 701 (p-STAT1) in response to IFN-gamma. STAT1 also typically promotes transcription of IFN-gamma. As a result of calcitriol treatment, p-STAT1 and IFN-gamma inhibition occurred while the vitamin D receptor (VDR), a nuclear receptor and transcription factor, increased on the protein level in TL-1 cells. We next sought to elucidate the mechanism behind calcitriol-mediated reduction in IFN-gamma production and whether VDR upregulation was required for this effect (Chapter 4). We found that calcitriol reduced IFN-gamma intracellular protein and mRNA transcript levels and p-STAT1 protein levels within 4 h. Moreover, calcitriol-mediated IFN-gamma reduction was independent of p-STAT1 levels but required VDR upregulation. Our results suggested that p-STAT1 and IFN-gamma levels were regulated independently of each other, indicating a dysregulation of the canonical IFN-gamma signaling pathway. Thus, in an effort to better understand the regulation of IFN-gamma, we sought to characterize the IFN-gamma-mediated signaling pathway from IFN-gamma signaling to transcription of IFN-gamma (Chapter 5). Previous studies demonstrated that T-LGLL cells have a deficiency in suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-gamma-mediated signaling. SOCS1 is typically induced in response to IFN-gamma, allowing for a tightly controlled signaling process. However, despite high IFN-gamma output, T-LGLL cells exhibit significantly lower SOCS1 levels compared to normal donor cells. Therefore, T-LGLL cells are likely to be unresponsive to IFN-gamma production, allowing an unchecked production of the inflammatory cytokine as seen in other cancers. We found that TL-1 cells have a significantly lower surface protein and mRNA transcript level of the IFN-gamma receptors (IFNGR) compared to Jurkat T cells, our positive IFN-gamma responsive cell line. IFN-gamma did not induce Janus kinase 2 (JAK2) or STAT1 phosphorylation or established IFN-gamma-mediated gene targets, including IRF-1 and SOCS1, in TL-1 cells. This further demonstrated a lack of responsiveness to IFN-gamma. We found that STAT5b, but not STAT1 or STAT3, played a role in regulating IFN-gamma transcript levels. Taken together, the decrease in IFNGR levels is a plausible explanation for the excessive IFN-gamma production and lack of negative regulation observed in T-LGLL. This pathway can be targeted effectively using calcitriol to significantly reduce IFN-gamma production. As calcitriol inhibits IFN-gamma independently of STAT1, calcitriol is able to reduce IFN-gamma regardless of its interactions with STAT1, providing a potent therapeutic to reduce inflammation. This thesis is the first study to evaluate IFN-gamma signaling and a treatment aimed at specifically targeting IFN-gamma in T-LGLL. Future studies are needed to assess the efficacy of calcitriol in T-LGLL patients in the clinic.

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