Resistance of glioblastoma-initiating cells to radiation mediated by the tumor microenvironment can be abolished by inhibiting transforming growth factor-ß.

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-ß (TGF-ß), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-ß type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-ß neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-ß per cell compared with conventional culture, suggesting that TGF-ß production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-ß inhibitors with radiation to improve therapeutic response in patients with GBM.

TGFß1 inhibition increases the radiosensitivity of breast cancer cells in vitro and promotes tumor control by radiation in vivo.

PURPOSE: To determine whether inhibition of TGFß signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: TGFß-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFß type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFß signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFß neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. RESULTS: Human breast cancer cell lines pretreated with TGFß small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFß growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFß type I receptor kinase inhibitor. Moreover, TGFß neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. CONCLUSION: These results show that TGFß inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy.

TGF-beta-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung.

Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-beta is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-beta in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-beta in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that O(2)-induced injury of the maturing lung is associated with TGF-beta-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-beta-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-beta signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-beta activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-beta antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-beta signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-beta antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung.

AAV2 vector harboring a liver-restricted promoter facilitates sustained expression of therapeutic levels of alpha-galactosidase A and the induction of immune tolerance in Fabry mice.

The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human alpha-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-alphagal). Moreover, AAV2/DC190-alphagal-mediated hepatic expression of alpha-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-alphagal-treated animals with recombinant human alpha-galactosidase A at 6 months failed to elicit the production of anti-alpha-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-alphagal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of alpha-galactosidase A to the liver may be a viable strategy for treating Fabry disease.

Equine infectious anaemia virus proteins with epitopes most frequently recognized by cytotoxic T lymphocytes from infected horses.

Efficacious lentiviral vaccines designed to induce cytotoxic T lymphocytes (CTL) in outbred populations with a diverse repertoire of MHC class I molecules should contain or express multiple viral proteins. To determine the equine infectious anaemia virus (EIAV) proteins with epitopes most frequently recognized by CTL from seven horses infected for 0.5 to 7 years, retroviral vector-transduced target cells expressing viral proteins were used in CTL assays. Gag p15 was recognized by CTL from 100% of these infected horses. p26 was recognized by CTL from 86%, SU and the middle third of Pol protein were each recognized by 43%, TM by 29%, and S2 by 14%. Based on these results, it is likely that a construct expressing the 359 amino acids constituting p15 and p26 would contain epitopes capable of stimulating CTL in most horses.