• Combinational immunotherapy of anti-OX40 antibody and IDO inhibitor synergistically enhances anti-tumor immune T cell-mediated response

      Berrong, Zuzana Jirina; Department of Biochemistry and Molecular Biology (2016-05)
      One of the major goals of cancer immunotherapy is to disrupt the immunosuppressive environment that allows tumors to thrive in and to generate potent and enduring antitumor specific-immune responses. Cancer vaccines may elicit antigen-specific immune responses; however, this is, in many tumor models and in human cancer, insufficient for positive outcomes due to existence of multiple immune-inhibitory mechanisms in tumors. A relatively recently introduced strategy to increase the therapeutic efficacy of tumor vaccination is to combine different immunological approaches that target different immunosuppressive pathways and to enhance the efficacy of vaccines by T cell agonists. OX40 is a co-stimulatory receptor expressed on T cells that can lead to proliferation and enhancement ofT cell effector function when bound by its ligand or targeted with agonist antibody. Here, we show that different doses of anti-OX40 antibody (Ab) elicit differential impacts on the T cell immune response resulting in either efficacious or detrimental therapeutic effect in immunized tumor-bearing mice. We demonstrate that treating tumor-bearing mice with an optimal dose of 1 mg/kg anti-OX40 Ab leads to a potent therapeutic and immune anti-tumor effect when combined with vaccine, whereas higher dose at 2.5 mg/kg of anti-OX40 Ab with vaccine increases the accumulation of regulatory T cells in the tumor and diminishes the therapeutic effect. Furthermore, we proposed that OX40 downstream molecular signaling through AKT activation in T cells may elucidate the differential T cell response when stimulated with anti-OX40 Ab. After optimizing the dose of agonist anti-OX40 Ab to stimulate the immune system toward maximal anti-tumor response when combined with vaccine, we strategized to improve the combinational therapy by targeting the so far untouched immunosuppressive environment. One of the immune suppressive molecules correlating with cancer progression is indoleamine-(2,3)-dioxygenase (IDO) enzyme. The catalytic activity of IDO hinders effector T cells from properly eliciting an anti-tumor effect. Here, we further evaluated the therapeutic outcome and immune mechanisms of the vaccineinduced immune response enhancement by agonist anti-OX40 antibody, while inhibiting the immunosuppressive IDO enzyme. We demonstrate that therapeutic efficacy of this combinational treatment leads to a profound inhibition of tumor growth and complete regression of established tumors in 60% of treated mice. We show that the mechanisms responsible for this therapeutic potency are: i) an increase in vaccine-induced tumorinfiltrating effector T cells that is facilitated by anti-OX40 antibody, and ii) a decrease of IDO enzyme activity within the tumor and the enhancement in the functionality of effector T cells that are facilitated by 1-methyl tryptophan (1-MT, IDO inhibitor). Our findings provide a promising and translatable strategy that can enhance the overall efficacy of cancer immunotherapy.
    • Deciphering mechanisms of DNA methylation regulation by depletion of the DNA methyltransferases and SETD2

      Tiedemann, Rochelle Lee; Department of Biochemistry and Molecular Biology / Cancer Center (2015)
      DNA methylation (5mC) is a stabile epigenetic mark that confers differential function for gene expression and chromatin accessibility dependent on the context and locality of the mark. Promoter regions populated by CpG islands (CGIs) are highly unmethylated while the remaining ∼80% of CpGs are methylated and distributed across gene bodies, repetitive and transposable elements, and intergenic regions of the genome. The presence and/or absence of particular histone modifications also dictate the patterning of 5mC genome-wide. In cancer, a reversal of 5mC patterns occur in which hypermethylation of tumor suppressor gene CGIs confers gene silencing, and hypomethylation of repetitive and transposable elements contribute to genomic instability. The mechanisms by which 5mC becomes aberrantly regulated in cancer remain unknown. In this study, direct and indirect mechanisms of 5mC regulation were investigated. To understand the direct regulation of 5mC genome-wide, we depleted cell line models of the DNA methyltransferases (DNMTs) that are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) 5mC patterns. Profiling of 5mC patterns on the Illumina HumanMethylation450 BeadChip revealed a unique antithetical relationship between DNMT1 and DNMT3B for the regulation of both 5mC and DNA hydroxymethylation (5hmC) across gene bodies. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCG versus CpG site methylation. DNMT3B depletion induced 5mC patterns that closely resemble those observed during cellular differentiation and occurred across gene bodies of highly expressed, H3K36me3-marked genes. SETD2, the histone methyltransferase responsible for H3K36me3 establishment across active gene bodies, was determined to influence the guidance of DNA methylation genome-wide through an indirect mechanism. SETD2 knockout induced widespread loss of H3K36me3 that did not coincide with changes in 5mC. However, paradoxical gains in H3K36me3 significantly induced hypermethylation and upregulation of underlying genes. Genes marked exclusively by the poised enhancer mark, H3K4me1, were commonly targeted for this epigenetic phenotype. DNA methylome profiling of loss-of-function SETD2 mutated clear cell renal cell carcinoma, papillary renal cell carcinoma, and lung adenocarcinoma tumors confirmed the predominance of the hypermethylation phenotype upon loss of SETD2. Collectively, these studies provide novel insight to understanding the regulatory mechanisms by which 5mC patterns are conferred.
    • Development of an Instrument to Measure the Appraisal of Cancer-related Fatigue

      Clark, Jane C.; Department of Biochemistry and Molecular Biology (2001-05)
      Based on a conceptual definition and model of fatigue, a three-phase descriptive study was conducted to develop items for a self-report instrument to measure the appraisal of fatigue. In Phase I, interviews with people experiencing cancer-related fatigue and a review of the literature were used to generate items to sample four constructs of the appraisal of fatigue: Fatigue, meaning, impact, and adaptability to fatigue. In Phase II, items were refined and reduced based on recommendations of a panel of content experts (N=7), instrument development expert, and pilot study results (N=20). In Phase III, reliability and validity estimates of the Fatigue Appraisal Scale were evaluated based on responses of a heterogeneous sample (N=196) of individuals diagnosed with cancer. Acceptable estimates of internal consistency reliability (Cronbach’s alpha) were determined for subscales of Fatigue (a =.91), Impact of Fatigue (a =.89), and Adaptability to Fatigue (a =.74). The internal consistency reliability of the Meaning of Fatigue subscale did not meet the required .60 for a new measurement instrument. To assess construct validity, hypotheses were generated and tested about the relationships of the subscales of the Fatigue Appraisal Scale and selected subscales of the Profile of Mood States-Shortened Form, Fatigue Assessment Instrument, and Functional Assessment of Cancer Therapy-Anemia. Results indicated low to moderate correlations (r = .20 to .70) among the subscales in the directions hypothesized with the exception of the correlation of the Adaptability to Fatigue subscale and the FACT-An Emotional and Functional Well-being subscales and the Impact of Fatigue subscale and the POMS Tension-anxiety subscale. Construct validity was examined further through factor analysis, using principal components analysis with varimax rotation. A four-factor solution with item loadings of .40 or greater on each factor was determined. Four items did not load on any of the four factors and were deleted. The factors were named: Global Fatigue, Impact of Fatigue, Adaptability to Fatigue, and Challenge of Fatigue. Items from the original Meaning of Fatigue subscale loaded on the Global Fatigue, Impact of Fatigue subscale or on the new factor, Challenge of Fatigue. Findings supported the factor structure for the instrument. Recommendations for future validation studies of the 34-item Revised Fatigue Appraisal Scale were offered.
    • Role of DNA methyltransferases in maintaining mammary stem/progenitor and cancer stem cells

      Pathania, Rajneesh; Department of Biochemistry and Molecular Biology (2015)
      Breast cancer is the leading cause of cancer death in women worldwide and it affects one in eight women in western countries. Like other human cancers, breast cancer also consists of cellular hierarchy, and heterogeneous. However, the cancer cell of origin and how a normal self-renewal pathway turns into abnormal self-renewal signaling are not known. DNA methylation provides a potential epigenetic mechanism for the cellular memory and heterogeneity, which needed to preserve the tumorigenic potential through repeated cell divisions. Further DNA methylation plays an essential role in stem/progenitor cell maintenance and provides a potential epigenetic mechanism for maintaining cellular memory and heterogeneity during self-renewal. However, the specific role of DNMTs in maintaining mammary stem cells (MaSC) and cancer stem cell (CSC) in a constantly replenishing organ, like mammary glands, is not yet known. Here, we show that Dnmt1 is essential for mammary gland development and indispensable for terminal end bud development and that mammary-gland specific Dnmt1 deletion in mice leads to significant reduction in mammary stem/progenitor cell formation. Moreover, Dnmt1 deletion almost completely abolishes Neu-Tg- and C3(1)-SV40-Tg- driven mammary tumor formation. The reduced tumor incidence observed in Dnmt1 deleted mouse is associated to significant reduction in cancer stem cell formation. These observations were recapitulated using pharmacological inhibitors of DNMTs in Neu-Tg mice in vivo. Further, we show that there is a substantial increase in DNMT1 expression when mammary stem/progenitor cells turn into tumor initiating cells. Using genome-scale methylation approach, we found that hypermethylation of genes involved in development and cell commitment pathways impart immortality and autonomous growth to the cancer stem cells. Moreover, our study provides evidence that stem cells, in addition to luminal progenitor cells, are susceptible for genetic and epigenetic modification and associated with chemotherapeutic resistance. Thus, combination of DNMT and HDAC inhibitors can be used as a therapeutic strategy to block mammary tumor formation and to overcome drug resistance by inhibiting CSCs. These findings improve our understanding of abnormal self-renewal associated with cell of origin, and highlight novel methylation markers that have the potential to serve as useful diagnostic tools and therapeutic targets in early detection of breast cancer.