Contributions
Abstract: EP855
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
Mantle cell Lymphoma (MCL) is a B cell tumor, characterized by frequent relapses. Chronic activation of multiple signaling pathways, including BTK, PI3K/AKT and NF-κB are of pathogenic importance in MCL. New drugs targeting the B Cell Receptor (BCR) dependent signaling pathways, such as ibrutinib, acalabrutinib, zanubrutinb and other BTK inhibitors, have been proved therapeutically efficient in MCL, but the disease remains uncurable. To this regard, there is an urgent need to discover novel druggable targets. We and others demonstrated that two Ser-Thr kinases, namely protein kinases CK1α and CK2, are essential kinases for the survival of multiple myeloma (MM) cell, acute myeloid leukemias, lymphomas and other hematological malignancies, regulating pivotal survival signaling events. We therefore sought to investigate a potential role of these kinases in MCL downstream the BCR signalling
Aims
In the present study we analyzed the role of CK1α and CK2, in the regulation of chronic active, BCR dependent signaling pathways in MCL, such as NF-κB, BTK and AKT, and the potential of their inhibition in association with the BTK inhibitor ibrutinib, to effectively reduce MCL cell growth and clonal expansion.
Methods
CK1α and CK2 expression and BCR signaling components were analyzed in MCL cells from patients, MCL cell lines and controls by western blot (WB). CK1α or CK2 silencing was obtained through the generation of anti-CK1α or anti CK2 shRNA IPTG-inducible MCL cell clones. CK1 or CK2 chemical inhibition was obtained with D4476 (CK1i) or CX-4945 (CK2i). Cell survival/apoptosis and proliferation were investigated with an array of standard techniques and the therapeutic interaction between drugs was analyzed through the Chou-Thalalay combination index method. CK2 knock down in vivo was obtained in xenograft NOD SCID mouse models.
Results
CK1α and CK2 are overexpressed in MCL cells compared to healthy B lymphocytes. CK1 or CK2 chemical inhibition with D4476 or CX-4945 respectively, caused, a reduction in the activating phosphorylation of S536 p65/RelA and S473 AKT, important signaling events downstream the BCR. CK1α or CK2 were shown to sustain MCL cell growth and proliferation, since their chemical inhibition or gene silencing caused MCL cell apoptosis and cell cycle arrest. We discover a novel, unanticipated potential role of these two kinases in regulating BTK function, since their chemical inhibition or silencing with RNAi, caused a reduction in the activating phosphorylation of BTK on Y223. We confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown. As a result, a striking cytotoxic effect on MCL cells was obtained through the combination of CK1α or CK2 inactivation with ibrutinib, as demonstrated by increased apoptotic Annexin V positive cells, PARP cleavage, reduction in pro-survival protein expression such as Mcl1 and pro-caspase 3. CK1i or CK2i acted synergically with ibrutinib, since the calculated combination index between the drugs (according to the Chou-Talalay methods) resulted less than 1,
Conclusion
Our findings suggest that CK1α and CK2 are fundamental kinases for MCL growth, regulating chronic active BCR-linked survival signaling cascades. Our data also indicate that these kinases could antagonize ibrutinib -induced apoptosis. Therefore, CK1α and CK2 could be rational therapeutic targets for the treatment of MCL, particularly those aggressive, ibrutinib-resistant forms.
Keyword(s): Ibrutinib, Kinase, Mantle cell lymphoma, Signal transduction
Abstract: EP855
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
Mantle cell Lymphoma (MCL) is a B cell tumor, characterized by frequent relapses. Chronic activation of multiple signaling pathways, including BTK, PI3K/AKT and NF-κB are of pathogenic importance in MCL. New drugs targeting the B Cell Receptor (BCR) dependent signaling pathways, such as ibrutinib, acalabrutinib, zanubrutinb and other BTK inhibitors, have been proved therapeutically efficient in MCL, but the disease remains uncurable. To this regard, there is an urgent need to discover novel druggable targets. We and others demonstrated that two Ser-Thr kinases, namely protein kinases CK1α and CK2, are essential kinases for the survival of multiple myeloma (MM) cell, acute myeloid leukemias, lymphomas and other hematological malignancies, regulating pivotal survival signaling events. We therefore sought to investigate a potential role of these kinases in MCL downstream the BCR signalling
Aims
In the present study we analyzed the role of CK1α and CK2, in the regulation of chronic active, BCR dependent signaling pathways in MCL, such as NF-κB, BTK and AKT, and the potential of their inhibition in association with the BTK inhibitor ibrutinib, to effectively reduce MCL cell growth and clonal expansion.
Methods
CK1α and CK2 expression and BCR signaling components were analyzed in MCL cells from patients, MCL cell lines and controls by western blot (WB). CK1α or CK2 silencing was obtained through the generation of anti-CK1α or anti CK2 shRNA IPTG-inducible MCL cell clones. CK1 or CK2 chemical inhibition was obtained with D4476 (CK1i) or CX-4945 (CK2i). Cell survival/apoptosis and proliferation were investigated with an array of standard techniques and the therapeutic interaction between drugs was analyzed through the Chou-Thalalay combination index method. CK2 knock down in vivo was obtained in xenograft NOD SCID mouse models.
Results
CK1α and CK2 are overexpressed in MCL cells compared to healthy B lymphocytes. CK1 or CK2 chemical inhibition with D4476 or CX-4945 respectively, caused, a reduction in the activating phosphorylation of S536 p65/RelA and S473 AKT, important signaling events downstream the BCR. CK1α or CK2 were shown to sustain MCL cell growth and proliferation, since their chemical inhibition or gene silencing caused MCL cell apoptosis and cell cycle arrest. We discover a novel, unanticipated potential role of these two kinases in regulating BTK function, since their chemical inhibition or silencing with RNAi, caused a reduction in the activating phosphorylation of BTK on Y223. We confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown. As a result, a striking cytotoxic effect on MCL cells was obtained through the combination of CK1α or CK2 inactivation with ibrutinib, as demonstrated by increased apoptotic Annexin V positive cells, PARP cleavage, reduction in pro-survival protein expression such as Mcl1 and pro-caspase 3. CK1i or CK2i acted synergically with ibrutinib, since the calculated combination index between the drugs (according to the Chou-Talalay methods) resulted less than 1,
Conclusion
Our findings suggest that CK1α and CK2 are fundamental kinases for MCL growth, regulating chronic active BCR-linked survival signaling cascades. Our data also indicate that these kinases could antagonize ibrutinib -induced apoptosis. Therefore, CK1α and CK2 could be rational therapeutic targets for the treatment of MCL, particularly those aggressive, ibrutinib-resistant forms.
Keyword(s): Ibrutinib, Kinase, Mantle cell lymphoma, Signal transduction