BRCAness as a biomarker of susceptibility to PARP inhibitors in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary brain cancer. GBMs commonly acquire resistance to standard-of-care therapies. Among the novel means to sensitize GBM to DNA-damaging therapies, a promising strategy is to combine them with inhibitors of the DNA damage repair (DDR) machinery, such as inhibitors for poly(ADP-ribose) polymerase (PARP). PARP inhibitors (PARPis) have already shown efficacy and have received regulatory approval for breast, ovarian, prostate, and pancreatic cancer treatment. In these cancer types, after PARPi administration, patients carrying specific mutations in the breast cancer 1 (BRCA1) and 2 (BRCA2) suppressor genes have shown better response when compared to wild-type carriers. Mutated BRCA genes are infrequent in GBM tumors, but their cells can carry other genetic alterations that lead to the same phenotype collectively referred to as ‘BRCAness’. The most promising biomarkers of BRCAness in GBM are related to isocitrate dehydrogenases 1 and 2 (IDH1/2), epidermal growth factor receptor (EGFR), phosphatase and tensin homolog (PTEN), MYC proto-oncogene, and estrogen receptors beta (ERβ). BRCAness status identified by accurate biomarkers can ultimately predict responsiveness to PARPi therapy, thereby allowing patient selection for personalized treatment. This review discusses potential biomarkers of BRCAness for a ‘precision medicine’ of GBM patients

Radiolabelled Bacillus anthracis toxin-based probes for molecular imaging of MMP activity in tumour models

Increased activity of matrix metalloproteinases (MMPs) is associated with poor prognosis and metastasis in different cancer types. To exploit this feature and target cancer cells, the protective antigen (PAWT) of the binary anthrax lethal toxin (LT) was modified to form pores in cell membranes only when cleaved by MMPs (PAL1). Anthrax lethal factor (LF) is then able to translocate through these pores into the cytosol of tumour cells. The aim of this work was to develop a novel non-invasive imaging agent for the detection of matrix metalloproteinase (MMP) activity based on engineered variants of LT. Here, 111In- radiolabelled form of LF was used with the PAL1/LF system to allow non-invasive in vivo imaging and quantification of MMP activity in tumour tissue by SPECT. Initially, PAL1 MMP-activation was tested in a cell free assay. Subsequently, the activation of PAL1 was evaluated by a cytotoxic assay in a range of cancer cell lines, with or without a broad spectrum MMP inhibitor. MMP2 and MMP9 activity in the different cancer cells was assessed by gelatin zymography. Expression levels of MMP14, and LT native receptors (TEM8 and CMG2) were evaluated by western blot analysis. LT components were radiolabelled with 111In. Cell abundance of TEM8 and CMG2 was determined by saturation binding assays using 111In-radiolabelled PAWT. After 111In-radiolabelling non-toxic variants of LF, their cell uptake was evaluated in the presence or not of PAL1. Evaluation of the in vivo pharmacokinetics and biodistribution of 111In-radiolabelled LT components was performed by SPECT/CT in naive mice or MMP-expressing MDA-MB-231 tumour-bearing mice. PAL1 MMP-cleavage was confirmed in a cell free assay. Notably, PAL1 capacity to cause cytotoxicity in a panel of cancer cells correlated with anthrax toxin receptor and MMP14 expression. Additionally, in the presence of MMP inhibitors PAL1 mediated cytotoxicity was prevented. Selective delivery of 111In-radiolabelled LF variants to MMP-expressing cells by PAL1 was demonstrated in vitro, and corroborated using confocal microscopy with fluorescently labelled variants of LF. Dynamic SPECT imaging demonstrated superior and selective uptake of radiolabelled PAL1 in tumour tissue when compared to radiolabelled PAWT. Finally, a radiolabelled LF variant was selectively delivered to MMP-positive MDA-MB-231 tumour bearing mice by PAL1, presenting excellent target-to-background contrast. Taken together, these results indicate that radiolabelled forms of mutated anthrax lethal toxin hold promise for non-invasive imaging of MMP activity in tumour tissue.</p

Estudo físicoquímico da associação do inibidor de serinoproteases BTCI com fluido magnético

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2012.Com o intuito de desenvolver novos materiais para tratamento do câncer, o objetivo deste trabalho foi associar dois componentes, sendo um com atividade anticarcinogênica comprovada: o BTCI e nanopartículas de maghemita recobertas por dextrana (MagDex) e caracterizar magnética e biofisicamente o complexo formado. A atividade antitumoral do BTCI foi comprovada contra células MCF-7 e essa molécula associada às nanopartículas biocompatíveis pode ser direcionada para um local sítio específico e lisar células tumorais no processo de magnetohipertermia. A caracterização do complexo foi feita pelos métodos: microscopia eletrônica de transmissão e de varredura, espalhamento de luz dinâmico (diâmetro hidrodinâmico, potencial zeta e polidispersividade), fluorescência (número de sítios e constante de associação), dicroismo circular, ensaios enzimáticos colorimétricos para avaliação da atividade inibitória do BTCI, birrefrigência magnética estática, ensaios de associação do inibidor. De acordo com análises de diâmetro hidrodinâmico, o BTCI complexou com as nanopartículas de MagDex. A constante de associação do complexo foi de 104 M-1 em pH 4,0 e 103 M-1 em pH 7,4 e pH 10,0. Segundo os parâmetros termodinâmicos, nessas três condições de pH, a associação do complexo foi espontânea, dirigida entropicamente com contribuições de associações hidrofóbicas e ligações de hidrogênio. A associação de BTCI a MagDex não alterou o conteúdo das estruturas secundárias, nem a atividade inibitória do BTCI complexado contra suas enzimas cognatas de forma expressiva. Adicionalmente, BTCI-MagDex manteve as características magnéticas de MagDex, como a maior resposta de susceptibilidade magnética a campos de (300 Oe), mesmo após diluições. O ensaio de sedimentação magnética sugere que 80% do BTCI associa às nanopartículas. Em conclusão, os resultados obtidos nesse trabalho sugerem que BTCI-MagDex apresenta características físico-químicas que favorecem a estabilidade e atividade do BTCI e a manutenção das propriedades magnéticas das nanopartículas, condição esta que é fundamental para estudos futuros que visem a aplicação desse sistema na terapia contra o câncer.In order to develop new materials for the treatment of cancer, the objective was to associate two components, one with proven anticancer activity: the BTCI and maghemite nanoparticles coated with dextran (MagDex) and magnetic and biophysically characterize the complex formed. The antitumor activity of BTCI was proven against MCF-7 cells and this molecule associated with biocompatible nanoparticles can be targeted to a specific site location and kill tumor cells in the process of magnetohipertermia. The characterization of the complex was made by the methods: transmission electron microscopy and scanning electron microscopy, dynamic light scattering (hydrodynamic diameter, zeta potential and polydispersity), fluorescence (number of sites and association constant), circular dichroism, enzymatic colorimetric assays for assessment inhibitory activity of BTCI, static magnetic birefringence, tests of association of the inhibitor. According to analysis of hydrodynamic diameter, BTCI formed a complex with MagDex nanoparticles. The association constant of the complex was 104 M-1 at pH 4.0 and 103 M-1 at pH 7.4 and pH 10.0. According to the thermodynamic parameters, these three pH conditions, the association of the complex was spontaneous, entropically driven with contributions from hydrophobic associations and hydrogen bonding. The association of BTCI-MagDex did not alter the content of secondary structures, nor the inhibitory activity of the complexed BTCI against their cognate enzymes significantly. Additionally, BTCI-MagDex kept MagDex magnetic characteristics, such as susceptibility response to magnetic fields (300 Oe), even after dilution. The test of magnetic sedimentation suggests that 80% of BTCI associated with nanoparticles. In conclusion, the results suggest that BTCI-MagDex has physicochemical characteristics that favor the stability and activity of BTCI and maintenance of the magnetic properties of nanoparticles, a condition which is critical for future studies aimed at applying this system in therapy against cancer

Radiolabelled Bacillus anthracis toxin-based probes for molecular imaging of MMP activity in tumour models

Increased activity of matrix metalloproteinases (MMPs) is associated with poor prognosis and metastasis in different cancer types. To exploit this feature and target cancer cells, the protective antigen (PAWT) of the binary anthrax lethal toxin (LT) was modified to form pores in cell membranes only when cleaved by MMPs (PAL1). Anthrax lethal factor (LF) is then able to translocate through these pores into the cytosol of tumour cells. The aim of this work was to develop a novel non-invasive imaging agent for the detection of matrix metalloproteinase (MMP) activity based on engineered variants of LT. Here, 111In- radiolabelled form of LF was used with the PAL1/LF system to allow non-invasive in vivo imaging and quantification of MMP activity in tumour tissue by SPECT. Initially, PAL1 MMP-activation was tested in a cell free assay. Subsequently, the activation of PAL1 was evaluated by a cytotoxic assay in a range of cancer cell lines, with or without a broad spectrum MMP inhibitor. MMP2 and MMP9 activity in the different cancer cells was assessed by gelatin zymography. Expression levels of MMP14, and LT native receptors (TEM8 and CMG2) were evaluated by western blot analysis. LT components were radiolabelled with 111In. Cell abundance of TEM8 and CMG2 was determined by saturation binding assays using 111In-radiolabelled PAWT. After 111In-radiolabelling non-toxic variants of LF, their cell uptake was evaluated in the presence or not of PAL1. Evaluation of the in vivo pharmacokinetics and biodistribution of 111In-radiolabelled LT components was performed by SPECT/CT in naive mice or MMP-expressing MDA-MB-231 tumour-bearing mice. PAL1 MMP-cleavage was confirmed in a cell free assay. Notably, PAL1 capacity to cause cytotoxicity in a panel of cancer cells correlated with anthrax toxin receptor and MMP14 expression. Additionally, in the presence of MMP inhibitors PAL1 mediated cytotoxicity was prevented. Selective delivery of 111In-radiolabelled LF variants to MMP-expressing cells by PAL1 was demonstrated in vitro, and corroborated using confocal microscopy with fluorescently labelled variants of LF. Dynamic SPECT imaging demonstrated superior and selective uptake of radiolabelled PAL1 in tumour tissue when compared to radiolabelled PAWT. Finally, a radiolabelled LF variant was selectively delivered to MMP-positive MDA-MB-231 tumour bearing mice by PAL1, presenting excellent target-to-background contrast. Taken together, these results indicate that radiolabelled forms of mutated anthrax lethal toxin hold promise for non-invasive imaging of MMP activity in tumour tissue.</p