355%, respectively), with equivalent tumour growth inhibition and ORR. lymphoma (DoHH-2, SuDHL-4), mantle cell lymphoma (Granta 519) and Burkitt’s lymphoma (RAMOS). Activity was also monitored in a systemic model of Granta 519. KEY RESULTS Navitoclax potentiated bendamustine activity in all cell lines tested. Bendamustine activated p53 in Granta 519 tumours, concurrent with activation of caspase 3. Navitoclax also improved responses to bendamustine-rituximab (BR) in a subset of tumours. CONCLUSIONS AND IMPLICATIONS Navitoclax in combination with bendamustine and BR is a viable combination strategy for use in the clinic and demonstrated superior efficacy compared with previously reported data for navitoclax plus CHOP and rituximab-CHOP. and (Kang in CD20-positive diffuse large B-cell lymphoma (DLBCL) and primary chronic lymphocytic leukaemia (CLL) lines (Chow in a Burkitt’s lymphoma (BL) model (Kanekal mice (DoHH-2, Granta 519) or C.B.-17 experiments for tumour growth inhibition and tumour growth delay were performed by Wilcoxon rank-sum analysis and KaplanCMeier log-rank analysis, respectively. Significance for %CR and %ORR was performed by two-tailed Fisher’s exact test. 0.05 Rgs5 versus vehicle or cytotoxic therapy (Wilcoxon rank-sum test for %TGI, KaplanCMeier log-rank analysis for %TGD). ? 0.05 versus vehicle or cytotoxic therapy (Fisher’s exact test). The combination of navitoclax and bendamustine had a greater than additive response in all three models tested. Tumour growth inhibition was significantly increased to 91%, 99% and 95% for DoHH-2, Granta 519 and RAMOS, respectively. Delay in tumour growth to 1 1 cm3 was also significantly enhanced by the Chlorothricin addition of navitoclax (146%, 355% and 93%, respectively). Importantly, ORR was significantly increased in all three lines as compared with bendamustine treatment alone, and Granta 519 also exhibited a significant increase in CR (Physique Chlorothricin 1, Table 1). Based on the substantial combination activity seen in Granta 519 tumours, this line was selected for further molecular analysis. Granta 519 tumours of approximately 500 mm3 were treated with bendamustine alone or in combination with navitoclax. Tumours were harvested at 4, 8 and 24 h and analysed by IHC for caspase induction; while changes in p53, Noxa and Mcl-1 were examined by Western blotting. Bendamustine treatment alone had little to no effect on caspase 3 cleavage Chlorothricin in Granta 519 tumours by IHC. However, addition of navitoclax induced a significant increase in cleaved caspase 3 staining at 24 h (Physique 2A). Protein levels of the tumour suppressor protein p53 increased at 24 h post treatment with bendamustine, with or without navitoclax co-treatment (Physique 2B). Expression of noxa, a p53-inducible BH3 protein that inhibits Mcl-1, was not altered by bendamustine treatment. Mcl-1 levels also remained unchanged at all time points. Increased p53 expression correlated temporally with the increased caspase 3 cleavage seen by IHC. Open in a separate window Physique 2 Molecular analysis of bendamustine in the absence and presence of navitoclax in Granta 519 flank tumours. Granta 519 tumours were treated with a single dose of bendamustine at 25 mgkg?1 with or without navitoclax at 100 mgkg?1. Tumours were harvested at 4, 8 and 24 h post treatment. Na?ve tumours were used as a control. (A) Immunohistochemical analysis of cleaved caspase 3. Tumours shown were harvested 24 h post therapy. CaLu-6 tumour treated with docetaxel was used as a positive control for staining. (B) Western blot analysis of expression levels of p53 and the Bcl-2 family members Noxa and Mcl-1. G519 lanes are Granta Chlorothricin 519 cells bortezomib as a Noxa control. Each lane represents an individual tumour. Rituximab further enhances the activity of bendamustine and bendamustine plus navitoclax in NHL tumours The addition of 10 mgkg?1 rituximab on the same day as bendamustine substantially enhanced bendamustine activity in DoHH-2 and RAMOS (Determine 3, Table 1). Strong tumour regressions were seen, with an ORR of 90% and 100%, respectively. Mice with RAMOS tumours had complete responses (CR = 80%) with BR treatment. In contrast, the addition of rituximab had little effect in the Granta 519 model, with no improvement in tumour growth inhibition, and only a minimal but significant increase in tumour growth delay (124% vs. 143%, respectively). Open in a separate window Physique 3 Xenograft sensitivity to treatment with navitoclax and BR. DoHH-2 (A), RAMOS (B) or Granta 519 (C) tumours were treated with triple vehicle, navitoclax for 14 days at 100 mgkg?1day?1,.