Contributions
Abstract: PB1850
Type: Publication Only
Background
Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by the accumulation of clonal B cells in peripheral blood, bone marrow and lymphoid tissues1. Recently, we demonstrated that two proteins endowed with cytoprotective activity, the Heat Shock Protein of 70kDa (HSP70) and the Heat Shock Factor 1 (HSF1), are overexpressed in CLL B cells and correlated to poor prognosis. HSF1 is regulated by a fine balance of activatory/inhibitory phosphorylations mediated by kinases belonging to RAS-triggered pathways2. Thanks to our previous study3, we hypothesize a model by which HSF1 is regulated through different RAS pathways, thus helping to gather information and dissect these networks in CLL. Particularly, the activation of RAS/PI3K/AKT is able to up-regulate HSP70 production, while the activation of the RAS/RAF/MEK/ERK pathway leads to the down-modulation of HSP70 expression.
Aims
Since we found that HSP70 and HSF1 were overexpressed in CLL and considering the pro-survival role of HSP70/HSF1 axis in cancer4, we are herein aimed at testing their druggability in CLL neoplastic B cells. In this context, we used molecules whose activity simultaneously affected the two RAS-mediated pathways, inhibiting AKT and activating ERK to the final purpose of down-modulating HSP70.
Methods
Freshly isolated leukemic B cells from therapy-free CLL patients were cultured in RPMI 1640 supplemented with antibiotics and 2% FBS and treated separately with: 40µM Resveratrol (a phenol); 10, 20 and 30µM Pterostilbene (a natural analogue of Resveratrol); 10, 50 and 100µM Triacetyl Resveratrol (a Resveratrol prodrug displaying superior bioavailability to Resveratrol); 5, 10 and 20µM Honokiol (a poly-phenolic compound whose action resemble that of Resveratrol). Apoptosis was evaluated after 24 hours by Annexin V/Propidium iodide flow cytometry test and by the presence of cleaved PARP in Western blotting.
Results
We found that in CLL, Resveratrol, or molecules with the same mechanism of action (i.e. inhibiting AKT activity and enhancing that of ERK) are able to induce apoptosis of neoplastic B cells in a dose-dependent manner. Particularly, we observed apoptosis after treatment with: 40µM Resveratrol (54±20% of living cells vs untreated cells, 70±18%; p<0.01, paired Student’s t Test); 10µM Triacetyl Resveratrol (65±8% of living cells vs untreated cells, 80±5%; p<0.01, paired Student’s t Test); 20µM Honokiol (26±29% of living cells vs untreated cells, 77±8%; p<0.05, paired Student’s t Test). Preliminary data on Pterostilbene show similar results.
Conclusion
HSP70 and HSF1 overexpression and correlation with poor prognosis in CLL patients underline their pivotal role in the regulation of leukemic B cell survival. For this reason, they represent interesting targets for anti-leukemic therapies. Of note, the use of molecules that simultaneously act at two different levels in the regulation of HSF1, and consequently of HSP70, should be considered in this field.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Heat shock protein
Abstract: PB1850
Type: Publication Only
Background
Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by the accumulation of clonal B cells in peripheral blood, bone marrow and lymphoid tissues1. Recently, we demonstrated that two proteins endowed with cytoprotective activity, the Heat Shock Protein of 70kDa (HSP70) and the Heat Shock Factor 1 (HSF1), are overexpressed in CLL B cells and correlated to poor prognosis. HSF1 is regulated by a fine balance of activatory/inhibitory phosphorylations mediated by kinases belonging to RAS-triggered pathways2. Thanks to our previous study3, we hypothesize a model by which HSF1 is regulated through different RAS pathways, thus helping to gather information and dissect these networks in CLL. Particularly, the activation of RAS/PI3K/AKT is able to up-regulate HSP70 production, while the activation of the RAS/RAF/MEK/ERK pathway leads to the down-modulation of HSP70 expression.
Aims
Since we found that HSP70 and HSF1 were overexpressed in CLL and considering the pro-survival role of HSP70/HSF1 axis in cancer4, we are herein aimed at testing their druggability in CLL neoplastic B cells. In this context, we used molecules whose activity simultaneously affected the two RAS-mediated pathways, inhibiting AKT and activating ERK to the final purpose of down-modulating HSP70.
Methods
Freshly isolated leukemic B cells from therapy-free CLL patients were cultured in RPMI 1640 supplemented with antibiotics and 2% FBS and treated separately with: 40µM Resveratrol (a phenol); 10, 20 and 30µM Pterostilbene (a natural analogue of Resveratrol); 10, 50 and 100µM Triacetyl Resveratrol (a Resveratrol prodrug displaying superior bioavailability to Resveratrol); 5, 10 and 20µM Honokiol (a poly-phenolic compound whose action resemble that of Resveratrol). Apoptosis was evaluated after 24 hours by Annexin V/Propidium iodide flow cytometry test and by the presence of cleaved PARP in Western blotting.
Results
We found that in CLL, Resveratrol, or molecules with the same mechanism of action (i.e. inhibiting AKT activity and enhancing that of ERK) are able to induce apoptosis of neoplastic B cells in a dose-dependent manner. Particularly, we observed apoptosis after treatment with: 40µM Resveratrol (54±20% of living cells vs untreated cells, 70±18%; p<0.01, paired Student’s t Test); 10µM Triacetyl Resveratrol (65±8% of living cells vs untreated cells, 80±5%; p<0.01, paired Student’s t Test); 20µM Honokiol (26±29% of living cells vs untreated cells, 77±8%; p<0.05, paired Student’s t Test). Preliminary data on Pterostilbene show similar results.
Conclusion
HSP70 and HSF1 overexpression and correlation with poor prognosis in CLL patients underline their pivotal role in the regulation of leukemic B cell survival. For this reason, they represent interesting targets for anti-leukemic therapies. Of note, the use of molecules that simultaneously act at two different levels in the regulation of HSF1, and consequently of HSP70, should be considered in this field.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Chronic Lymphocytic Leukemia, Heat shock protein