Expression and Activity of the NF-κB Subunits in Chronic Lymphocytic Leukaemia: A Role for RelB and Non-Canonical Signalling.

BACKGROUND: Canonical NF-κB signalling by p65 (RelA) confers chemo-resistance and poor survival in chronic lymphocytic leukaemia (CLL). The role of non-canonical NF-κB signalling (leading to RelB and p52 subunit activation) in CLL is less understood, but given its importance in other B-cell tumour types, we theorised that RelB and p52 may also contribute to the pathology of CLL. METHODS: DNA binding activity of all five NF-kB subunits, p65, p50, RelB, p52, and c-Rel, was quantified using ELISA and correlated to ex vivo chemoresistance, CD40L-stimulated signalling (to mimic the lymph node microenvironment), and clinical data. RESULTS: Importantly, we show for the first time that high basal levels of RelB DNA binding correlate with nuclear RelB protein expression and are associated with del(11q), ATM dysfunction, unmutated IGHV genes, and shorter survival. High levels of nuclear p65 are prevalent in del(17p) cases (including treatment-naïve patients) and also correlate with the outcome. CD40L-stimulation resulted in rapid RelB activation, phosphorylation and processing of p100, and subsequent CLL cell proliferation. CONCLUSIONS: These data highlight a role for RelB in driving CLL cell tumour growth in a subset of patients and therefore strategies designed to inhibit non-canonical NF-κB signalling represent a novel approach that will have therapeutic benefit in CLL.

PARP1 expression, activity and ex vivo sensitivity to the PARP inhibitor, talazoparib (BMN 673), in chronic lymphocytic leukaemia.

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors.We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 - 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 - 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2'-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function.PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib.

Therapeutic potential of poly(ADP-ribose) polymerase inhibitor AG014699 in human cancers with mutated or methylated BRCA1 or BRCA2.

BACKGROUND: Mutations in BRCA1 and BRCA2 (BRCA1/2), components of the homologous recombination DNA repair (HRR) pathway, are associated with hereditary breast and ovarian cancers. Poly(ADP-ribose) polymerase (PARP) inhibitors are selectively cytotoxic to animal cells with defective HRR, but results in human cancer cells have been contradictory. We undertook, to our knowledge, the first comprehensive in vitro and in vivo investigations of the antitumor activity of the PARP inhibitor AG014699 in human cancer cells carrying mutated or epigenetically silenced BRCA1/2. METHODS: We used nine human cell lines, four with nonmutated BRCA1/2 (MCF7, MDA-MB-231, and HCC1937-BRCA1 [breast cancer] and OSEC-2 [ovarian surface epithelial]), two with mutated BRCA1 (MDA-MB-436 and HCC1937 [breast cancer]), one with mutated BRCA2 (CAPAN-1 [pancreatic cancer]), one that was heterozygous for BRCA2 (OSEC-1 [ovarian surface epithelial]), and one with epigenetically silenced BRCA1 (UACC3199 [breast cancer]), and two Chinese hamster ovary cell lines, parental AA8 and XRCC3 mutated IRS 1SF. We assessed cytotoxicity, DNA damage, and HRR function. Antitumor activity of AG014699 was determined by growth of xenograft tumors (five mice per treatment group). Long-term safety of AG014699 was assessed. RESULTS: AG014699 (≤10 µM) was cytotoxic to cells with mutated BRCA1/2 or XRCC3 and to UACC3199 cells with epigenetically silenced BRCA1 but not to cells without BRCA1/2 or XRCC3 mutations or that were heterozygous for BRCA2 mutation. AG014699 induced DNA double-strand breaks in all nine cell lines studied. HRR was observed only in cells with functional BRCA1/2 proteins. Growth of xenograft tumors with BRCA1/2 mutations or with epigenetically silenced BRCA1 was reduced by AG014699 treatment, and combination treatment with AG014699 plus carboplatin was more effective than either drug alone. AG014699 was not toxic in mice with nonmutated or heterozygous BRCA2. CONCLUSION: Human cancer cells or xenograft tumors with mutated or epigenetically silenced BRCA1/2 were sensitive to AG014699 monotherapy, indicating a potential role for PARP inhibitors in sporadic human cancers.

6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance.

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

Characterisation of adult green lacewing (Chrysoperla carnea) digestive physiology: impact of a cysteine protease inhibitor and a synthetic pyrethroid.

BACKGROUND: In spite of concern regarding potential non-target effects of GM crops, few studies have compared GM pest control with conventional methods. The impacts of cypermethrin and oilseed rape expressing oryzacystatin-1 (OC-1) were compared in this study on the predator Chrysoperla carnea (Stephens). RESULTS: Adults fed purified rOC-1 showed a subtle shift in digestive protease profile, with an increasing reliance on serine proteases (chymotrypsin), increase in aspartic proteases and a slight reduction in elastase activity. Although there were no effects on mortality, onset of oviposition was delayed; however, once egg production commenced, egg laying and hatching success rates were comparable with those of controls. Oryzacystatin-1 expressed in pollen showed no detrimental effects. Cypermethrin had no effect on mortality owing to high levels of non-specific esterase activity resulting in partial breakdown of the insecticide. In spite of this, there was a significant delay in onset of oviposition and a significant reduction in egg production and viability. CONCLUSION: This study demonstrates the potential for pest management to impact on predators, but importantly it highlights the ability of the predator to detoxify/respond to treatments with different modes of action. In this case, exposure to an insecticide carried a greater fitness cost than exposure to a protease inhibitor expressed in transgenic crops.

Inhibition of poly(ADP-ribose) polymerase-1 enhances temozolomide and topotecan activity against childhood neuroblastoma.

PURPOSE: High-risk neuroblastoma is characterized by poor survival rates, and the development of improved therapeutic approaches is a priority. Temozolomide and topotecan show promising clinical activity against neuroblastoma. Poly(ADP-ribose) polymerase-1 (PARP-1) promotes DNA repair and cell survival following genotoxic insult; we postulated that its inhibition may enhance the efficacy of these DNA-damaging drugs in pediatric cancers. EXPERIMENTAL DESIGN: We evaluated the chemosensitizing properties of the PARP inhibitor AG014699 (Pfizer, Inc.) in combination with temozolomide and topotecan, against human neuroblastoma cells and xenografts, alongside associated pharmacologic and toxicologic indices. RESULTS: Addition of PARP-inhibitory concentrations of AG014699 significantly potentiated growth inhibition by both topotecan (1.5- to 2.3-fold) and temozolomide (3- to 10-fold) in vitro, with equivalent effects confirmed in clonogenic assays. In two independent in vivo models (NB1691 and SHSY5Y xenografts), temozolomide caused a xenograft growth delay, which was enhanced by co-administration of AG014699, and resulted in complete and sustained tumor regression in the majority (6 of 10; 60%) of cases. Evidence of enhanced growth delay by topotecan/AG014699 co-administration was observed in NB1691 xenografts. AG014699 metabolites distributed rapidly into the plasma (Cmax, 1.2-1.9 nmol/L at 30 min) and accumulated in xenograft tissues (Cmax, 1-2 micromol/L at 120 min), associated with a sustained suppression of PARP-1 enzyme activity. Doses of AG014699 required for potentiation were not toxic per se. CONCLUSIONS: These data show enhancement of temozolomide and topotecan efficacy by PARP inhibition in neuroblastoma. Coupled with the acceptable pharmacokinetic, pharmacodynamic, and toxicity profiles of AG014699, our findings provide strong rationale for investigation of PARP inhibitors in pediatric early clinical studies.

Effective sensitization of temozolomide by ABT-888 is lost with development of temozolomide resistance in glioblastoma xenograft lines.

Resistance to temozolomide and radiotherapy is a major problem for patients with glioblastoma but may be overcome using the poly(ADP-ribose) polymerase inhibitor ABT-888. Using two primary glioblastoma xenografts, the efficacy of ABT-888 combined with radiotherapy and/or temozolomide was evaluated. Treatment with ABT-888 combined with temozolomide resulted in significant survival prolongation (GBM12: 55.1%, P = 0.005; GBM22: 54.4%, P = 0.043). ABT-888 had no effect with radiotherapy alone but significantly enhanced survival in GBM12 when combined with concurrent radiotherapy/temozolomide. With multicycle therapy, ABT-888 further extended the survival benefit of temozolomide in the inherently sensitive GBM12 and GBM22 xenograft lines. However, after in vivo selection for temozolomide resistance, the derivative GBM12TMZ and GBM22TMZ lines were no longer sensitized by ABT-888 in combination with temozolomide, and a similar lack of efficacy was observed in two other temozolomide-resistant tumor lines. Thus, the sensitizing effects of ABT-888 were limited to tumor lines that have not been previously exposed to temozolomide, and these results suggest that patients with newly diagnosed glioblastoma may be more likely to respond to combined temozolomide/poly(ADP-ribose) polymerase inhibitor therapy than patients with recurrent disease.

Bitrophic and tritrophic effects of Bt Cry3A transgenic potato on beneficial, non-target, beetles.

Insect-resistant transgenic plants have been suggested to have unpredictable effects on the biodiversity of the agro-ecosystem, including potential effects on insect natural enemies, beneficial in control of crop pests. Whilst carnivorous as adults, many of these predators may also consume plant tissues, in particular plant pollen and nectar. Coleoptera are important in terms of agro-ecological research not only because of the large number of species in this order, but also because of their role as biological control agents. Thus any detrimental impact on this group of insects would be highly undesirable. The effects of potato expressing the coleopteran-specific Bacillus thuringiensis delta-endotoxin Cry3A (Bt Cry3A) on the ladybird beetle Harmonia axyridis and the carabid beetle Nebria brevicollis were investigated via the bitrophic interaction of the adult ladybird with potato flowers and the tritrophic interaction of the carabid consuming a non-target potato pest. Immunoassays confirmed accumulation of the transgene product in potato leaves and floral tissues (at levels of up to 0.01% (pollen) and 0.0285% (anthers) of total soluble protein). Despite H. axyridis and N. brevicollis belonging to the targeted insect order, no significant effects upon survival or overall body mass change of either beetle were observed. Furthermore, Bt Cry3A had no detrimental effects on reproductive fitness of either beetle species, either in terms of fecundity or subsequent egg viability. Behavioural analysis revealed no significant impact of Bt Cry3A on beetle activity or locomoter behaviour. Ligand blots indicate that this is due to either the absence of Bt-binding sites in brush border membrane vesicles (BBMV) isolated from Nebria brevicollis, or in the case of Harmonia axyridis, the binding did not functionally lead to behavioural or physical effects.

Prey-mediated effects of transgenic canola on a beneficial, non-target, carabid beetle.

Transgenic plants producing insecticidal proteins from Bacillus thuringiensis (Bt) can control some major insect pests and reduce reliance on sprayed insecticides. However, large scale adoption of this technology has raised concerns about potential negative effects, including evolution of pest resistance to Bt toxins, transgene flow from Bt crops to other plants, and harm to non-target beneficial organisms. Furthermore, concern has also been expressed over the effects this technology may have on biodiversity in general. Ecologically relevant risk assessment is therefore required (Risk = Hazard x Exposure). Transgenic plants that produce Bt toxins to kill insect pests could harm beneficial predators. This might occur directly by transmission of toxin via prey, or indirectly by toxin-induced reduction in prey quality (Hazard). To test these hypotheses, we determined the effects of Bt-producing canola on a predatory ground beetle (Pterostichus madidus) fed larvae of diamondback moth (Plutella xylostella) that were either susceptible or resistant to the Bt toxin. Survival, weight gain, and adult reproductive fitness did not differ between beetles fed prey reared on Bt-producing plants and those fed prey from control plants. Furthermore, while Bt-resistant prey was shown to deliver high levels of toxin to the beetle when they were consumed, no significant impact upon the beetle was observed. Subsequent investigation showed that in choice tests (Exposure), starved and partially satiated female beetles avoided Bt-fed susceptible prey, but not Bt-fed resistant prey. However, in the rare cases when starved females initially selected Bt-fed susceptible prey, they rapidly rejected them after beginning to feed. This prey type was shown to provide sufficient nutrition to support reproduction in the bioassay suggesting that Bt-fed susceptible prey is acceptable in the absence of alternative prey, however adults possess a discrimination ability based on prey quality. These results suggest that the direct effects of Bt-producing canola on predator life history was minimal, and that predators' behavioural preferences may mitigate negative indirect effects of reduced quality of prey caused by consumption of Bt-producing plants. The results presented here therefore suggest that cultivation of Bt canola may lead to conservation of non-target predatory and scavenging organisms beneficial in pest control, such as carabids, and may therefore provide more sustainable agricultural systems than current practices. In addition, minimal impacts on beneficial carabids in agro-ecosystems suggest that Bt canola crops are likely to be compatible with integrated pest management (IPM) systems.

Comparing the impact of conventional pesticide and use of a transgenic pest-resistant crop on the beneficial carabid beetle Pterostichus melanarius.

The potential impact of a chemical pesticide control method has been compared with that of transgenic plants expressing a protease inhibitor conferring insect resistance by utilising a tritrophic system comprising the crop plant Brassica napus (L.) (Oilseed rape), the pest mollusc Deroceras reticulatum (Müller) and the predatory carabid beetle Pterostichus melanarius (Illiger). Cypermethrin, as the most widely used pesticide in UK oilseed rape (OSR) cultivation, was selected as the conventional treatment. OSR expressing a cysteine protease inhibitor, oryzacystatin-1 (OC-1), was the transgenic comparator. In feeding trials, D. reticulatum showed no significant long-term effects on measured life history parameters (survival, weight gain, food consumption) as a result of exposure to either the cypermethrin or OC-1 treatment. However, D. reticulatum was able to respond to the presence of the dietary inhibitor by producing two novel proteases following exposure to OC-1-expressing OSR. Similarly, P. melanarius showed no detectable alterations in mortality, weight gain or food consumption when feeding on D. reticulatum previously fed either pesticide-contaminated or GM plant material. Furthermore, as with the slug, a novel form of protease, approximately M(r) 27 kDa, was induced in the carabid in response to feeding on slugs fed OC-1-expressing OSR.