Oncology clinical trials and insurance coverage: An update in a tenuous insurance landscape

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151873/1/cncr32360_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151873/2/cncr32360.pd

Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 (APE1/Ref-1) redox function negatively regulates NRF2

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications

Hypoxia-Inducible Factor-1α Regulates CD55 in Airway Epithelium

Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium

Allergic airway disease in mice alters T and B cell responses during an acute respiratory poxvirus infection.

Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8(+) T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8(+) T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4(+) T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism

Blocking IL-10R signaling in VV-infected AAD mice resulted in altered disease severity.

<p>VV-infected mice were treated with Rat IgG1 control mAb or αIL-10R blocking mAb at 3 (i.p.), 4 (i.n.) and 6 dpi (i.p.), and these animals were sacrificed on day 9. (A) Lung VV titers and (B) bronchiole inflammation were not altered by IL-10R mAb blockade. Blocking IL-10R signaling increased (C) levels of VV-specific IgG1 in serum, (D) IFNγ protein levels in BAL fluid and (E) the frequency of infiltrating CD4⁺ IFNγ⁺ T cells in the lungs of VV-infected AAD mice. Treatment with an αIL-10R mAb did not alter the infiltration of (F) CD8⁺ IFNγ⁺, (H) CD4⁺ IL-10⁺ or (I) CD8⁺ IL-10⁺ T cells, butincreased recruitment of (J) CD4⁺ PD-1⁺ T cells in the lungs of VV-infected AAD mice. Blocking IL-10R significantly decreased (G) BAL IL-10 protein secretion and significantly increased (K) BAL T cells but not (L) total BAL cells in VV-infected mice with AAD. Statistical significance was determined by a One-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001 αIL-10R mAb vs. IgG1. Results are expressed as the mean ± SEM for 3–5 mice in each group and are representative of 2 independent experiments.</p

Induction of AAD and pulmonary VV inoculation altered expression of cytokines in the lungs.

<p>Relative expression of gene transcripts in lung tissue was measured using qRT-PCR. Transcripts for pro-allergic cytokines (A) <i>Il13</i>, (B) <i>Il17a</i> and (C) <i>Il5</i>, but not (D) <i>Il6</i>, were increased in AAD and VV-infected AAD mice. Transcripts for (E) <i>Il10</i> and (F) <i>Ifng</i> were elevated in VV-infected mice and VV-infected AAD mice. In VV-infected mice, (G) IL-10 and (H) IFNγ secretion in BAL fluid peaked by 9 dpi, as measured by ELISA. AAD mice inoculated with VV secreted more IL-10 and IFNγ at 9 dpi compared to non-allergic mice infected with VV. Data are expressed as the mean ± SEM for four mice in each group. Data are representative of 2 independent experiments. Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 AAD+VV vs. control; ^#<i>P</i><0.05, ^##<i>P</i><0.01 AAD+VV vs. VV; ⁺⁺⁺<i>P</i><0.001, ⁺⁺⁺⁺<i>P</i><0.0001 AAD+VV vs. AAD.</p

Preexisting AAD exacerbated pulmonary VV infection.

<p>(A) AAD was induced in mice by repeated OVA i.p. sensitizations and respiratory challenges over a course of 19 days. The resulting mice with AAD or control mice were inoculated at day 21 with 10⁴ PFU VV i.t. followed by monitoring for virus-induced pathology. VV titer and weight loss profiles from two separate cohorts of treated animals are shown. (B, D) Virus titers were measured in homogenized lung tissue using a viral plaque assay as described in the methods. Viral persistence and titer were significantly higher in AAD mice 10–12 dpi. The largest difference in virus titer between the VV mice and VV-infected AAD mice was observed at 10 dpi. (C, E) Mice were weighed starting one day after VV infection and the percent weight change was normalized to this day. The kinetics of weight loss after VV infection was not altered by AAD, but the maximal weight loss was significantly increased in AAD+VV mice. All values are represented as mean ± SEM, 4–14 mice per group. (D) Statistical significance was determined by a One-way ANOVA: ^###<i>P</i><0.001 AAD+VV vs. VV. (B, C, E) Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, ***<i>P</i><0.001, ****<i>P</i><0.0001 AAD+VV vs. control; ^#<i>P</i><0.05, ^##<i>P<0.01</i>, ^###<i>P</i><0.001 AAD+VV vs. VV. The following abbreviations are used in all figure legends: VV, vaccinia virus; AAD, allergic airway disease; AAD+VV, allergic airway disease+vaccinia virus. The cohort of mice examined in panels B and C were also used in experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g002" target="_blank">Figures 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g005" target="_blank">5</a>. The cohort of mice examined in panels D and E were also used in experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g006" target="_blank">Figures 6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062222#pone-0062222-g007" target="_blank">7</a>.</p

AAD caused increased epithelium disruption and cellular hyperplasia regardless of VV infection.

<p>Murine lung tissue was fixed in 10% formalin and paraffin-embedded sections were stained with PAS/hematoxylin and blindly scored for several pathophysiological parameters using a semi-quantitative scale of 0–3. Mice with AAD had (A) increased bronchiole epithelium disruption (yellow arrow) in the airways, (B) increased perivascular lymphoid hyperplasia (yellow inverted triangle), and (C) increased giant cell pneumonia (red diamond-headed arrow) at 2 and 9 dpi. (D) Goblet cell hyperplasia (yellow box) was significantly increased in AAD+VV mice at 2 dpi, but significantly decreased at 12 dpi compared to AAD mice. (E–G) PAS/hematoxylin-stained slides were digitally imaged with the Aperio Scan Scope CS system at 20× magnification. Multifocal necrotizing pneumonia is evident in the AAD+VV mice at 9 and 12 dpi as diffuse pink staining in the lung parenchyma (red arrow). Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: NS - not significant, ⁺<i>P</i><0.05, ⁺⁺<i>P</i><0.01 ⁺⁺⁺<i>P</i><0.001 AAD+VV vs. AAD.</p

VV-infected AAD mice had increased signs of airway inflammation.

<p>(A) Non-invasive plethysmography was used to assess animal breathing. This analysis revealed increased responses to methacholine challenge (Penh) for control, VV and AAD mice at day 7 post-inoculation or mock treatment. In contrast, AAD mice inoculated with VV had elevated baseline Penh measurements suggesting an altered breathing pattern which was not sensitive to methacholine exposure. (B) Murine lung tissue was fixed in 10% formalin and paraffin-embedded sections were stained with H&E. Lung tissue inflammation was assessed by light microscopy and blindly scored using a semi-quantitative scale of 0–4, with a measure of 0 reflecting no inflammation, and 4 indicative of severe inflammation of peribronchiolar, periarterial and parenchymal spaces. AAD mice with or without VV infection had severe bronchiolar inflammation 2 dpi. VV-infected AAD mice had sustained inflammation through day 12 compared to AAD mice. (C) AAD mice with or without VV infection had elevated inflammatory cell infiltration in the BAL at 2 dpi. VV-infected mice with AAD had prolonged inflammatory cell infiltration in the BAL through 9 dpi. All values represented as mean ± SEM, 4–14 mice per group and representative of 3 independent experiments. Statistical significance was determined by a Two-way ANOVA with Bonferroni’s multiple comparisons test: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 AAD+VV vs. control; ^#<i>P</i><0.05, AAD+VV vs. VV. ⁺⁺⁺<i>P</i><0.001 AAD+VV vs. AAD.</p

VV-infected AAD mice had increased CD8⁺ effector T cells in the lungs at 10 dpi.

<p>Lung tissue was harvested from mice 10 days after virus or mock inoculation. Single cell suspensions from individual animals were restimulated in vitro as described in the methods followed by antibody staining and flow cytometric analyses. The frequency of (A) CD4⁺ and CD8⁺, (B) IL-10⁺ (C) CD4⁺ IFNγ⁺, (D) CD4⁺ IL-10⁺, (E) CD8⁺ IFNγ⁺ and (F) CD8⁺ IL-10⁺ infiltrating lung T cells was determined by cell counts and FACS analysis using commercial antibodies as outlined in the methods. (G–H) Epitope-specific CD8⁺ T cells were determined by MHC I tetramer staining for H-2 class I epitopes (D^d: E3 epitope; K^d: A52 epitope; L^d: F2 epitope) and FACS analysis (white bar E3, gray bar A52, black bar F2). Cells from dissociated lung tissue were restimulated in vitro and tetramer-stained as detailed in the methods. (G) Ratio of epitope-specific CD8⁺ T cells normalized to total CD8⁺ tetramer⁺ T cells. (H) Frequency of epitope-specific CD8⁺ T cells with or without IFNγ co-expression was determined. Statistical significance was determined by a One-way ANOVA with Bonferroni’s multiple comparisons test *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ****<i>P</i><0.0001 AAD+VV vs. control; ^#<i>P</i><0.05, ^###<i>P</i><0.001 AAD+VV vs. VV. Results are expressed as mean ± SEM for (A–F) six mice in each group and are representative of 2 independent experiments or (G–H) 4–5 mice in each group.</p