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Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms.

Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, Vannucchi AM - J. Cell. Mol. Med. (2013)

Bottom Line: We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated.Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models.In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

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Combination of BEZ235 and ruxolitinib synergistically inhibits JAK2V617F mutated Ba/F3-EPOR and SET2 cells, EEC from PV patients, and CD34+-derived colonies from PMF patients. (A–C) Progressively increasing amounts of BEZ235 and ruxolitinib were used according to the Chou and Talalay method to estimate the interactions of the drugs in cultures of Ba/F3-EPOR VF cells (A) and SET2 cells (B), and in semisolid media favouring the growth of EEC progenitors from four PV patients (C). End-points were the measure of proliferation inhibition in cultures of Ba/F3-EPOR VF and SET2 cells and the number of EPO-independent erythroid colonies in the EEC assay. A combination Index (CI) was then calculated; a CI < 0.9 indicates that the interaction of the two drugs is synergistic. ‘Fa’ is the cell fraction affected by combined amounts of the two drugs. (D) The effects of combination of BEZ235 and ruxolitinib was also measured in clonogenic assay using CD34+ cells obtained from three patients with PMF. Erythroid and myeloid colonies were counted together to calculate the cell fraction affected (only Fa50 and Fa80 values are shown).
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fig03: Combination of BEZ235 and ruxolitinib synergistically inhibits JAK2V617F mutated Ba/F3-EPOR and SET2 cells, EEC from PV patients, and CD34+-derived colonies from PMF patients. (A–C) Progressively increasing amounts of BEZ235 and ruxolitinib were used according to the Chou and Talalay method to estimate the interactions of the drugs in cultures of Ba/F3-EPOR VF cells (A) and SET2 cells (B), and in semisolid media favouring the growth of EEC progenitors from four PV patients (C). End-points were the measure of proliferation inhibition in cultures of Ba/F3-EPOR VF and SET2 cells and the number of EPO-independent erythroid colonies in the EEC assay. A combination Index (CI) was then calculated; a CI < 0.9 indicates that the interaction of the two drugs is synergistic. ‘Fa’ is the cell fraction affected by combined amounts of the two drugs. (D) The effects of combination of BEZ235 and ruxolitinib was also measured in clonogenic assay using CD34+ cells obtained from three patients with PMF. Erythroid and myeloid colonies were counted together to calculate the cell fraction affected (only Fa50 and Fa80 values are shown).

Mentions: Previous works 18–39 indicated that the JAK2 inhibitor ruxolitinib inhibited the proliferation and induced apoptosis of JAK2V617F mutated cells lines. Therefore, we evaluated the effects of co-treatment of ruxolitinib and BEZ235 on the viability of Ba/F3-EPOR VF and SET2 cell lines; to determine potential synergy, we calculated the CI according to Chou and Talalay 37. As shown in Figure 3A, the calculated CI at varying drug combinations indicated strong synergistic activity in the two cell lines. For example, a 50% inhibition of Ba/F3-EPOR VF cell proliferation was obtained at 30 nM BEZ235 and 80 nM ruxolitinib compared with 87 nM and 220 nM, respectively, when the drugs were used alone (Fig. 3A). In case of SET2 cells, 50% inhibition of cell proliferation was obtained using 55 nM BEZ235 and 26 nM ruxolitinib, compared with 334 and 160 nM with each drug alone (Fig. 3B). We next determined the effects of co-treatment of BEZ235 and ruxolitinib in an EEC assay (n = 4 PV patients). A 50% inhibition of EEC colony formation was observed at 4.4 nM BEZ235 and 0.4 nM ruxolitinib compared with 20 and 1.8 nM, respectively, for the two drugs alone (Fig. 3C). Efficacy of drug combination was also analysed by measuring the inhibition of GFU-GM and BFU-E colony formation by CD34+ cells of three PMF individuals. As shown in Figure 3D, the combination of BEZ235 and ruxolitinib resulted in synergistic inhibition of colony formation compared with single agents. We finally analysed by western blot the level of phosphorylated mTOR, 4EBP1 and STAT5 in Ba/F3-EPOR VF cells exposed to BEZ235 and ruxolitinib alone and in combination (Fig. 4). We found that drug combination produced greater inhibition of phosphorylated mTOR, 4EBP1 and particularly STAT5 compared with single drugs; of note, no significant changes were observed in Ba/F3-EPOR wt cells at the doses employed.


Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms.

Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, Vannucchi AM - J. Cell. Mol. Med. (2013)

Combination of BEZ235 and ruxolitinib synergistically inhibits JAK2V617F mutated Ba/F3-EPOR and SET2 cells, EEC from PV patients, and CD34+-derived colonies from PMF patients. (A–C) Progressively increasing amounts of BEZ235 and ruxolitinib were used according to the Chou and Talalay method to estimate the interactions of the drugs in cultures of Ba/F3-EPOR VF cells (A) and SET2 cells (B), and in semisolid media favouring the growth of EEC progenitors from four PV patients (C). End-points were the measure of proliferation inhibition in cultures of Ba/F3-EPOR VF and SET2 cells and the number of EPO-independent erythroid colonies in the EEC assay. A combination Index (CI) was then calculated; a CI < 0.9 indicates that the interaction of the two drugs is synergistic. ‘Fa’ is the cell fraction affected by combined amounts of the two drugs. (D) The effects of combination of BEZ235 and ruxolitinib was also measured in clonogenic assay using CD34+ cells obtained from three patients with PMF. Erythroid and myeloid colonies were counted together to calculate the cell fraction affected (only Fa50 and Fa80 values are shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig03: Combination of BEZ235 and ruxolitinib synergistically inhibits JAK2V617F mutated Ba/F3-EPOR and SET2 cells, EEC from PV patients, and CD34+-derived colonies from PMF patients. (A–C) Progressively increasing amounts of BEZ235 and ruxolitinib were used according to the Chou and Talalay method to estimate the interactions of the drugs in cultures of Ba/F3-EPOR VF cells (A) and SET2 cells (B), and in semisolid media favouring the growth of EEC progenitors from four PV patients (C). End-points were the measure of proliferation inhibition in cultures of Ba/F3-EPOR VF and SET2 cells and the number of EPO-independent erythroid colonies in the EEC assay. A combination Index (CI) was then calculated; a CI < 0.9 indicates that the interaction of the two drugs is synergistic. ‘Fa’ is the cell fraction affected by combined amounts of the two drugs. (D) The effects of combination of BEZ235 and ruxolitinib was also measured in clonogenic assay using CD34+ cells obtained from three patients with PMF. Erythroid and myeloid colonies were counted together to calculate the cell fraction affected (only Fa50 and Fa80 values are shown).
Mentions: Previous works 18–39 indicated that the JAK2 inhibitor ruxolitinib inhibited the proliferation and induced apoptosis of JAK2V617F mutated cells lines. Therefore, we evaluated the effects of co-treatment of ruxolitinib and BEZ235 on the viability of Ba/F3-EPOR VF and SET2 cell lines; to determine potential synergy, we calculated the CI according to Chou and Talalay 37. As shown in Figure 3A, the calculated CI at varying drug combinations indicated strong synergistic activity in the two cell lines. For example, a 50% inhibition of Ba/F3-EPOR VF cell proliferation was obtained at 30 nM BEZ235 and 80 nM ruxolitinib compared with 87 nM and 220 nM, respectively, when the drugs were used alone (Fig. 3A). In case of SET2 cells, 50% inhibition of cell proliferation was obtained using 55 nM BEZ235 and 26 nM ruxolitinib, compared with 334 and 160 nM with each drug alone (Fig. 3B). We next determined the effects of co-treatment of BEZ235 and ruxolitinib in an EEC assay (n = 4 PV patients). A 50% inhibition of EEC colony formation was observed at 4.4 nM BEZ235 and 0.4 nM ruxolitinib compared with 20 and 1.8 nM, respectively, for the two drugs alone (Fig. 3C). Efficacy of drug combination was also analysed by measuring the inhibition of GFU-GM and BFU-E colony formation by CD34+ cells of three PMF individuals. As shown in Figure 3D, the combination of BEZ235 and ruxolitinib resulted in synergistic inhibition of colony formation compared with single agents. We finally analysed by western blot the level of phosphorylated mTOR, 4EBP1 and STAT5 in Ba/F3-EPOR VF cells exposed to BEZ235 and ruxolitinib alone and in combination (Fig. 4). We found that drug combination produced greater inhibition of phosphorylated mTOR, 4EBP1 and particularly STAT5 compared with single drugs; of note, no significant changes were observed in Ba/F3-EPOR wt cells at the doses employed.

Bottom Line: We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated.Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models.In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Show MeSH
Related in: MedlinePlus