PF527 PROTEIN KINASE CK1 ALPHA INACTIVATION CAUSES MANTLE CELL LYMPHOMA GROWTH ARREST AND SYNERGISTICALLY ENHANCES IBRUTINIB CYTOTOXICITY
Authors: Manni S. , Carrino M. , Fregnani A. , Tubi L. Quotti , Visentin A. , Trentin L. , Semenzato G. , Piazza F. 
Published: June 2019

Background:

Mantle Cell lymphoma (MCL) is a B‐cell malignancy comprising roughly 5–10% of B non Hodgkin lymphomas. MCL patients have been demonstrated to be particularly sensitive to the Bruton Tyrosin kinase (BTK) inhibitor ibrutinib, which by impinging on B Cell Receptor (BCR)‐associated signalling events, causes neoplastic B cell apoptosis and proliferation arrest. Nevertheless, refractoriness to ibrutinib may develop portending a dismal prognosis, thus for such cases novel therapeutic strategies are urgently needed. We recently demonstrated that protein kinase CK1α is pivotal for multiple myeloma (MM) cell growth and its inactivation empowers bortezomib and lenalidomide MM induced cytotoxicity. The known CK1α‐dependent regulation of growth‐promoting cascades, such as the NF‐κB and AKT in other tumors, suggests a potential role in MCL downstream the BCR signalling.

Aims:

In this study we analyzed the role of CK1α on MCL survival and ibrutinib cytotoxicity. We asked whether CK1α takes part in ibrutinib‐induced MCL cell apoptosis and whether blocking CK1α with the chemical inhibitor D4476, or through RNA interference could empower ibrutinib mediated cytotoxicity, through the deregulation of important BCR related survival signalling cascades such as NF‐κB and AKT.

Methods:

CK1α expression and BCR/ NF‐κB signalling components were analyzed in MCL patients’ cells, in cell lines and controls by western blot (WB) or immunofluorescence. CK1α silencing was obtained through the generation of anti‐CK1α shRNA IPTG‐inducible MCL cell clones or through electroporation of double strand CK1α‐directed siRNA in MCL cell lines. CK1α chemical inhibition was obtained with the compound D4476. Cell survival/apoptosis and proliferation were investigated with Annexin V/Propidium Iodide labelling and cytofluorimetric analysis, analysis of PARP cleavage and of pro‐apoptotic/pro‐survival proteins expression in WB. The combination index between ibrutinib and D4476 was calculated through the MTT assay according to the Chou Talalay method

Results:

Inhibiting CK1α in MCL cells with D4476 or RNAi reduced cell viability, determined apoptosis and proliferation arrest as judged by increased PARP cleavage and caspase activity, by a reduced expression of the pro‐survival Mcl1 protein and by deregulation of the cell cycle. CK1α inhibition empowered ibrutinib induced reduction of activating phosphorylation of RelA/p65 in Ser 536 and of AKT in Ser 473, without affecting ERK1/2 activation. CK1 inactivation together with ibrutinib boosted ibrutinib induced cytotoxicity and resulted in a synergic anti‐lymphoma effect with a calculated combination index inferior than 1.

Summary/Conclusion:

Our findings suggest that CK1α sustains MCL growth through the regulation of BCR‐linked survival signalling cascades and protects from ibrutinib‐induced apoptosis. CK1 inhibition could represent a rational therapeutic option for MCL and offer the groundwork to design novel combination treatments for this disease.