Kathryn Bondra

Polyanhydride nanovaccine against swine influenza virus in pigs.

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4+CD8αα+ T helper and CD8+ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs

ABSTRACT Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross‐protection against evolving viruses in the field. Poly(lactic‐co‐glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA‐KAg) were prepared, which were spherical in shape with 200 to 300 nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA‐KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T‐helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA‐KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA‐KAg vaccinated pig lung airways were observed. Immunologically, PLGA‐KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN‐&ggr; secreting total T cells, T‐helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA‐NPs reduced the clinical disease and induced cross‐protective cell‐mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans.

Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma.

Curative therapy for childhood sarcoma presents challenges when complete resection is not possible. Ionizing radiation (XRT) is used as a standard modality at diagnosis or recurrence for childhood sarcoma; however, local recurrence is still problematic. Most childhood sarcomas are TP53 wild type at diagnosis, although approximately 5–10% have MDM2 amplification or overexpression.

Using NanoDot dosimetry to study the RS 2000 X-ray Biological Irradiator

Abstract Purpose: To use NanoDot dosimeters to study the RS 2000 X-ray Biological Irradiator dosimetry characteristics and perform in vivo dosimetry for cell or small animal experiments. Methods and materials: We first calibrated the Landauer NanoDot™ Reader by irradiating some NanoDot dosimeters with a set of known doses at specific positions defined by the irradiator. A group of five NanoDot dosimeters were placed at five specific positions where the dose rates were known and provided by the irradiator. Each group was irradiated for a set of times respectively. By correlating the readings of dosimeters with the given irradiated doses, we established the dose-reading relationship for the irradiator under the specific running condition. The established calibration curve was validated by exposing arbitrary known doses to a set of dosimeters, using the Landauer NanoDot™ Reader to measure the doses, and then making the comparison between the two doses. To study the dose gradient of the X-ray inside the irradiated target (dose variation/cm), we placed dosimeters under different thicknesses of water-equivalent bolus and irradiated them, then measured the doses to determine the dose gradient. Results: Using the method described above, we were able to calibrate the Landauer InLight NanoDot™ Reader and use NanoDot dosimeters to measure the actual doses delivered to the targets for the cell/small animal experiments that use the RS 2000 X-ray Biological Irradiator. Conclusions: NanoDots are ideal dosimeters to use for in vivo dosimetry for cell/small animal irradiation experiments. The dose decrease inside the animal tissue is about 20% per cm.

Radiation therapy may increase metastatic potential in alveolar rhabdomyosarcoma

We previously determined that radiation could be safely administered using a mouse‐flank in vivo model to both alveolar (Rh30) and embryonal (Rh18) rhabdomyosarcoma xenografts. Mice from both tumor lines in this experiment developed metastases, an event not previously described with these models. We sought to determine if radiation‐induced changes in gene expression underlie an increase in the metastatic behavior of these tumor models.

Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma

Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low‐grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long‐term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF‐mutant anaplastic astrocytoma.

FANCD2 is a potential therapeutic target and biomarker in alveolar rhabdomyosarcoma harboring the PAX3-FOXO1 fusion gene.

Purpose: Alveolar rhabdomyosarcoma that harbors the PAX3–FOXO1 fusion gene (t-ARMS) is a common and lethal subtype of this childhood malignancy. Improvement in clinical outcomes in this disease is predicated upon the identification of novel therapeutic targets. Experimental Design: Robust mouse models were used for in vivo analysis, and molecular studies were performed on xenografts treated in parallel. Two independent patient sets (n = 101 and 124) of clinically annotated tumor specimens were used for analysis of FANCD2 levels and its association with clinical and molecular characteristics and outcomes. Results: Our xenograft studies reveal a selective suppression of FANCD2 by m-TOR kinase inhibition and radiosensitization of the t-ARMS line only. In the initial patient set, we show that FANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the copresence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and nonrandom enrichment of mTOR-associated genes that correlate with FANCD2 gene expression within the t-ARMS samples, but not within other cases. In the second patient set, we show that on a protein level, FANCD2 expression correlates with PAX3–FOXO1 fusion gene and is strongly associated with phospho-P70S6K expression in cases with the fusion gene. Conclusions: Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS. Indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy. Clin Cancer Res; 20(14); 3884–95. ©2014 AACR.

The Application of Radiation Therapy to the Pediatric Preclinical Testing Program (PPTP): Results of a Pilot Study in Rhabdomyosarcoma

The Pediatric Preclinical Testing Program (PPTP) has been successfully used to determine the efficacy of novel agents against solid tumors by testing them within a mouse‐flank in vivo model. To date, radiation therapy has not been applied to this system. We report on the feasibility and biologic outcomes of a pilot study using alveolar and embryonal rhabdomyosarcoma xenograft lines.

Abstract 477: Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Patients with recurrent low-grade brain tumors or anaplastic astrocytoma may receive radiation treatment, however, the long-term sequellae from radiation treatment can be severe. For pediatric astrocytomas, low-grade tumors are associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion (Grade 1) or through an activating point mutation of BRAF (predominantly V600E; Grade 2-4) and is the most common lesion in low- and intermediate-grade astrocytoma. Previous studies have indicated synergy between MEK inhibition and ionizing radiation therapy (XRT), although the proposed mechanisms for synergy differ. We have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic childhood astrocytoma. The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant (V600E) glioblastoma cell lines (DBTRG-05MG, AM380c1). The effect of MEK inhibition (selumetinib, AZD6244, ARRY-142886), XRT (2 Gy daily fractionated doses), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. Inhibition of MEK signaling by selumetinib, suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of TORC1 signaling and decreased levels of FANCD2 protein, whereas inhibition of TORC1 signaling was less in DBTRG-05MG cells and even less in AM380c1 cells. The level of TORC1 inhibition by selumetinib was associated with loss of clonogenic survival, and suppression of FANCD2 in vitro. In vivo we examined the sensitivity of BT-40 xenografts to selumetinib (75 mg/kg BID x 42 days, PO), XRT (10 Gy delivered as 2 Gy daily fractions), or selumetinib combined with XRT. For control tumors, median time to event was 17 days, whereas for XRT alone it was 80 days (P<0.0001). Selumetinib treatment induced complete regression of most tumors with progression starting around week 5 of the 6-week treatment period, consistent with previous data, with median time to event being 51 days (P<0.0001 vs control). In contrast to XRT or selumetinib as single agents, the combination caused complete regression of all tumors, with only 4 of 10 (40%) recurrent tumors within the 24 weeks observation period, and significantly better than radiation or selumetinib alone (P<0.0001 for both comparisons). These data add to studies that indicate synergy between XRT and MEK inhibitors, and suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF. Combining MEK inhibition with XRT may allow maintenance of tumor control at lower doses of XRT that would decrease long-term sequellae for children with BRAF-mutant brain tumors. Citation Format: Adam W. Studebaker, Kathryn Bondra, Star Seum, Justin Leasure, Christopher Chronowski, Changxian Shen, Doris Phelps, Paul D. Smith, Raushan T. Kurmasheva, Xiaokui Mo, Peter J. Houghton. Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 477. doi:10.1158/1538-7445.AM2015-477

Abstract 1614: Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma

Curative therapy for childhood sarcoma presents challenges when complete resection is not possible. Ionizing radiation (XRT) is used as a standard modality at diagnosis or recurrence for childhood sarcoma, however local recurrence is still problematic. Most childhood sarcomas are TP53 wild type at diagnosis, and approximately 5-10 per cent have MDM2 amplification or overexpression. Thus, reconstitution of a functional TP53 pathway, through inhibition of MDM2-mediated TP53 degradation, is an attractive anticancer strategy to enhance the activity of cytotoxic therapies that induce apoptosis through TP53-dependent pathways. RG7388, is a second generation MDM2 inhibitor. However, combining MDM2 inhibitors with myelosuppressive therapies may be problematic as the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis suggests that thrombocytopenia may be an on-target toxicity. Here we have evaluated the antitumor activity of RG7388 alone or in combination with XRT in two rhabdomyosarcoma xenografts (Rh18 [embryonal], and Rh30 [alveolar]). RG7388 was administered at 80 mg/kg daily x 5 (schedule 1) or 100 mg/kg BID q7 days x 2 (schedule 2). XRT (2 Gy daily fractions) was given to a cumulative dose of 20 Gy (Rh18) or 30 Gy (Rh30). XRT (20 Gy) induced complete regressions of Rh18 xenografts followed by regrowth of all tumors with the median event time of 89.6 days compared 9.5 days for control or RG7388 treated tumors (P = 0.0287). The combination of XRT with RG7388 on either schedule induced complete regressions with no tumor regrowth (19 weeks observation). Thus, RG7388 given on either schedule significantly potentiated XRT (P Tumor samples were derived from untreated tumors, or tumors following 2, 4 or 6 Gy XRT and 24 and 48 Hr after the last XRT fraction with or without RG7388, or at the same time points from mice treated for 3 days with RG7388 alone (80 mg/kg). RG7388/XRT increased p21 levels over the 3 days of treatment, to a greater extent than XRT alone whereas p21 induction in tumors treated with RG7388 alone was lowest. Both XRT and combination treatments induced PARP cleavage over the first 48 hr. Although additional models should be examined to see whether the synergistic activity of XRT combined with RG7388 occurs frequently, these initial results suggest that the combination may provide more effective local control for pediatric soft tissue sarcoma. Citation Format: Peter J. Houghton, Doris A. Phelps, Kathryn Bondra, Star Seum, Christopher Chronowski, Justin Leasure, Raushan T. Kurmasheva, Stephen Middleton, Dian Wang, Xiaokui Mo. Inhibition of MDM2 by RG7388 confers hypersensitivity to X-radiation in xenograft models of childhood sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2015-1614