Cyclin-dependent kinase 5 (CDK5) contributes to Bruton tyrosine kinase inhibitor (BTKI) resistance via IRE1alpha/Xbp1s axis in mantle cell lymphoma (MCL)

Resistance to BTKI is inevitable in MCL. While it may be partially driven by BTK mutations, full mechanisms are not understood. Here we evaluated the role of CDK5 in acquired BTKI resistance. We generated ibrutinib-resistant (IR) MCL cell lines (JeKo and Mino). RNA-seq of JeKo-IR cells showed that CDK5 was one of the upregulated genes with a fold change of 2.15 vs. parental cells, and findings were confirmed by IB. CDK5 is a proline serine/threonine protein kinase that modulates cell cycle proteins in neuronal tissue, however its role in cancer is not known. We established CDK5 overexpression (OE; by sgRNA) in JeKo and Mino cells. CDK5-OE cells exhibited enhanced proliferation and partial resistance to ibrutinib. By contrast, knockdown (KD; shRNA) of CDK5 in JeKo-IR and Mino-IR cell lines re-sensitized cells to ibrutinib. Treatment with GFB-12811, a selective CDK5 inhibitor, reduced proliferation of IR and CDK5-OE MCL cell lines in a dose dependent manner. We next evaluated CDK5 expression following activation of B-cell receptor signaling. IgM and BAFF crosslinking resulted in rapid increase in BTK phosphorylation which was accompanied by upregulated CDK5 protein levels. Furthermore, primary MCL cells cultured in BAFF- or CD40L-expressing stromal conditions (which induce ibrutinib resistance) upregulated CDK5. NSG mice xenografted with CDK5-OE JeKo cells exhibited inferior survival compared with mice xenografted with control cells. Mass spectrometry analysis of cell lines with manipulated CDK5 revealed >1000 differentially expressed proteins and >300 phosphosites, with cell cycle and metabolism-related pathways affected by CDK5. Kinase activity profiling revealed that 29 kinases were differentially active in CDK5-altered cells. In particular, Src kinase activation directly correlated with CDK5 expression. These findings were supported by RNA-seq and validated by immunoblotting. Furthermore, GSEA analysis of RNA-Seq revealed upregulation of the Unfolded Protein Response (UPR), Inflammatory response, Myc targets, p53 pathway and OxPhos in JeKo-IR cells. We next focused on the UPR. Both IR and CDK5-OE JeKo and Mino cells exhibited increased phosphorylation of IRE1alpha at Ser724 residue and upregulation of Xbp1, compared with control cell lines. By contrast, both pIRE1alpha and Xbp1 were decreased following CDK5-KD. Immunoprecipitation demonstrated a novel direct interaction between CDK5 and IRE1alpha. Finally, Xbp1-KD in JeKo-IR and Mino-IR cells led to reduced proliferation and viability, comparable to cells with CDK5-KD. In sum, CDK5 is overexpressed in MCL cells with acquired resistance to BTKi. Genetic or pharmacologic targeting CDK5 partially abrogates ibrutinib resistance. CDK5 contributes to BTKi resistance via modulation of the XBP1/IRE1alpha axis of the UPR pathway. Thus, CDK5 is a potential therapeutic target in MCL.