DOI

10.17077/etd.4d8c6hku

Document Type

Dissertation

Date of Degree

Spring 2018

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular and Cell Biology

First Advisor

Fenghuang Zhan

First Committee Member

John D. Colgan

Second Committee Member

Michael D. Henry

Third Committee Member

Dawn E. Quelle

Fourth Committee Member

Hasem Habelhah

Abstract

Multiple Myeloma (MM) is an incurable plasma cell malignancy and, although novel treatment regimes in the past decade have improved patient outcome, long-term treatment leads to relapse and refractory disease. The centrosomal kinase NEK2 is found overexpressed in MM and promotes chromosomal instability, drug resistance and increased proliferation. Although much research shows NEK2 having a detrimental effect in cancer, much of its mechanisms of overexpression and drug resistance has not been studied in detail. In this work we expand our understanding of NEK2 in MM.

Using Tandem Affinity Purification coupled with Mass Spectrometry, we show that NEK2 directly interacts with the de-ubiquitinase USP7. We confirm this interaction in cell lines of MM and lung cancer. Since USP7 has been shown to have important cancer-promoting roles we tested if USP7 was necessary for NEK2-driven bortezomib resistance. We found that USP7 shRNA was sufficient to sensitize the bortezomib resistant NEK2 overexpressing cells to bortezomib. Surprisingly, we found that USP7 inhibition with shRNA or by treatment with the small molecule USP7 inhibitor P5091 led to depletion of NEK2 protein in every cell line tested. Previous research shows USP7’s main function is a de-ubiquitinase and, since NEK2 is a target of the ubiquitin-proteasome system, we hypothesized USP7 may be de-ubiquitinating NEK2. Through western blots and immunoprecipitations, we show the NEK2-USP7 interaction promotes the de-ubiquitination and subsequent stabilization of NEK2, presenting USP7 as the first discovered de-ubiquitinating enzyme of NEK2. To understand how NEK2 promotes drug resistance in cancer we studied a previously published list of NEK2-regulated genes and, using the UCSC genome browser (Track Name:GM12878+TNFa RELA) ChIP-seq data, we found approximately half of these genes have the NF-κB transcription factor p65 bound throughout the gene sequence. We also produced a signaling score using an average of 11 known targets of NF-κB and patients with high NEK2 showed a significantly increased score of NF-κB signaling. Additionally, through western blots and immunofluorescence, we found that patients with high NEK2 protein levels consisitently had activation higher signal of p65 protein and phosphorylated p65 at Serine 536, indicative of increased activity. We then causally show NEK2 activates canonical NF-κB by performing western blots and a dual-luciferase reporter assay on control and NEK2 overexpressing cells. Using AKT and PP1α inhibitors, we found that NEK2 drives NF-κB by phosphorylating and inactivating PP1α, leading to hyperactive AKT. Using this model of NEK2-NF-κB activation, we aimed at targeting NEK2 directly with the small molecule drugs INH1 (depletes NEK2 protein) and P5091 (inhibits USP7 activity) in empty vector control cells, NEK2 overexpressing cells or cells with an acquired drug resistance phenotype. Our results show that both INH1 and P5091 can overcome bortezomib resistance in cell lines and in vivo.

Another aspect of MM disease we targeted in this work was bone disease. Bone disease in MM is common and causes bone pain and fractures but a much is still regarding what drives these lesions. We found that NEK2 expression in patients correlates with a presence of bone lesions, based on FDG-PET scan and MRI. Using our previously published list of NEK2 regulated genes, we found Heparanase (HPSE) is directly correlated to NEK2 expression. HPSE is an extracellular protein shown to promote differentiation of the bone destroying cell, osteoclast. Using western blots, RT-qPCR and ELISA, we found NEK2 increases HPSE expression and extracellular release. HPSE was also on the list of genes upregulated by NEK2 found to have p65 bound to the gene, thus we tested if NEK2 was driving HPSE through the NF-κB. Accordingly, we found NEK2 drives HPSE through the NF-κB pathway and, consistent with our previous results, in a USP7-dependent manner. Using bone marrow macrophages and conditioned media from empty vector control or NEK2 overexpressing cells, we found NEK2 promtoes increased differentiation of osteoclasts and inhibition of HPSE blocked this effect, strongly suggesting HPSE is the mediator of this effects. Importantly these findings were recapitulated in vivo. Empty vector or NEK2 overexpressing cells were injected through the tail vein to allow dissemination to the bone marrow. microCT and Xray revealed mice injected with NEK2 overexpressing cells showed reduced bone density, compared to empty vector cells. Additionally, H&E and TRAP staining confirmed our in vitro results by showing higher osteoclast levels in bone sections of mice injected with NEK2 overexpressing cells.

Lastly, we show a novel role for the ATPase TRIP13 as a cofactor for USP7 de-ubiquitinating activity. TRIP13 is overexpressed in cancer, has been shown to be an oncogene and promotes drug resistance. By systematically targeting TRIP13 overexpressing cells with drugs that inhibit different pathways we found TRIP13 drug resistance is diminished by inhibiting USP7. We found that TRIP13 binds with USP7 and by western blots and immunoprecipitations we show it is necessary for the de-ubiquitination of NEK2. Furthermore, we also found TRIP13 shows a hyperactive USP7 phenotype, shuttling PTEN out of the nucleus and stabilizing MDM2, in a USP7 dependent manner.

In summary, this work shows the de-ubiquitinase USP7, coupled with the ATPase TRIP13 stabilizes NEK2 by de-ubiquitination, this leads to accumulation of NEK2 and activation of the canonical NF-κB pathway through PP1α/AKT, which promotes drug resistance and activates HPSE, increasing osteoclast differentiation and bone destruction.

Keywords

Cancer, Drug resistance, Multiple Myeloma, NEK2, osteoclasts, USP7

Pages

xix, 140 pages

Bibliography

Includes bibliographical references (pages 128-140).

Comments

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Copyright

Copyright © 2018 Reinaldo Franqui Machin

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