The Interaction of NEK2 with USP7 Causes Resistance to Proteasome Inhibitor in Multiple Myeloma

Reinaldo Franqui-Machin; Mu Hao; Bai Hua; Zhimin Gu; Xin Zhan; Hongwei Xu; Hasem Habelhah; Yogesh Jethava; Lugui Qiu; Ivana Frech; Guido J Tricot; Fenghuang Zhan

doi:10.1182/blood.V130.Suppl_1.593.593

Background: Drug resistance is the major reason for treatment failure in multiple myeloma (MM). Our previous work has demonstrated that high expression of chromosomal instability (CIN) genes results in increased cell survival and drug resistance and is associated with poor outcome in MM and other cancers. NEK2 was the most significant CIN gene to induce drug resistance and poor outcome in MM. Though we have demonstrated that NEK2 induces drug resistance through activation of efflux drug pumps, the detail mechanisms remain unknown. Materials and Methods: Multiple MM cell lines, xenograft MM NOD-Rag1nullmice with both subcutaneous and by tail vein injection, and primary MM samples were included in this study to test NEK2 inhibitors and gene expression profiles (GEP). The tandem affinity purification - mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were applied for the identification of NEK2 interacting partners. The dual-luciferase reporter assay and GEPs were performed to determine whether the interaction of NEK2 with USP7 activates the canonical NF-kB signaling pathway. Chromatin immunoprecipitation (ChIP)-qPCR and western blots were carried out to determine the direct target genes of the axis NEK2/USP7/NF-kB. Results: We demonstrate that NEK2 is stabilized by the de-ubiquitinating enzyme USP7, leading to accumulation of NEK2 within the cancer cells. Accumulation of NEK2 activates the canonical NF-kB through the AKT/IKK/IKB-a/p65 axis and, using this pathway as framework, we uncovered a novel bona fide NF-kB target, heparanase, a protein that is known to promote bone destruction in MM patients. A NF-kB or USP7 inhibitor could overcome NEK2-induced bortezomib resistance. The NEK2 inhibitor, INH1, showed great therapeutic effects on MM in vitro and in vivo . To corroborate our findings, we subcutaneously injected NEK2-OE ARP1 MM cells into NOD.Cg-Rag1 mice and targeted NEK2 with the small molecule drugs INH1 or P5091 (USP7 inhibitor) alone or in combination with bortezomib. As expected bortezomib had very little effect in reducing tumor burden since NEK2-OE cells are highly resistant to this drug. Inhibition of NEK2 with P5091 or INH1 alone had a strong anti-tumor effect and a synergistic effect was observed when combined with bortezomib. Conclusion: Our data present a novel mechanism for the interaction of NEK2 with USP7 driving drug resistance in MM. We conclude that (1) USP7 in MM cells leads to the de-ubiquitination and escape of proteasomal degradation of NEK2, leading to its accumulation in cancer cells. (2) Accumulated NEK2 binds and phosphorylates PP1a, suppressing its phosphatase activity thus promoting AKT overactivation. (3) AKT then phosphorylates the IKKa/b complex, triggering the canonical NF-kB cascade which phosphorylates and targets IKBa for proteasomal degradation, releasing the sequestered p65 to be phosphorylated at S536 and translocation to the nucleus where it drives bortezomib resistance by activating its target genes. Our data also show that INH1 and P5091 can deplete NEK2 protein in vitro and in vivo and suppress NEK2 downstream targets resulting in overcoming bortezomib resistance. Our findings should facilitate novel targeted approaches to the therapy and prevention of myeloma progression and relapse. No relevant conflicts of interest to declare.

The Interaction of NEK2 with USP7 Causes Resistance to Proteasome Inhibitor in Multiple Myeloma

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