Dissertation
Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2018
DOI: 10.17077/etd.4d8c6hku
Abstract
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
Details
- Title: Subtitle
- Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma
- Creators
- Reinaldo Franqui Machin - University of Iowa
- Contributors
- Fenghuang Zhan (Advisor)John D. Colgan (Committee Member)Michael D. Henry (Committee Member)Dawn E. Quelle (Committee Member)Hasem Habelhah (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Molecular and Cellular Biology
- Date degree season
- Spring 2018
- DOI
- 10.17077/etd.4d8c6hku
- Publisher
- University of Iowa
- Number of pages
- xix, 140 pages
- Copyright
- Copyright © 2018 Reinaldo Franqui Machin
- Comment
This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/.
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 128-140).
- Public Abstract (ETD)
Multiple Myeloma (MM) is an incurable blood cancer. In the last 10 years treatments for MM have greatly increased survival rates, however patients eventually relapse and become unresponsive to treatment. Thus, understanding drug resistance in MM is of upmost importance to eradicate this disease.
NEK2 is a protein accumulated in MM and other cancers. Cells with higher NEK2 levels are resistant to anti-MM drugs, however the mechanisms that lead to its accumulation and drug resistance are still unknown. In this thesis, we show that NEK2 protein is stabilized by interacting with the protein USP7. This USP7-dependant stabilization of NEK2 leads to its accumulation and subsequent activation of the pro-survival pathway, NF-kB. We show that suppressing NF-kB can reverse NEK2-mediated drug resistance, suggesting NF-kB is important for the drug resistance acquired by NEK2 overexpression. Our work also shows that MM patients with high NEK2 have increased bone lesions, which cause bone pain and fractures. We found that NEK2, through NF-kB, promotes bone damage by activating the drug resistance and metastasis driver, Heparanase (HPSE).
Lastly, our work unveils the genetic instability protein, TRIP13, as a novel cofactor vital for USP7’s activity. We show that, without TRIP13 expression, USP7 becomes inactive, failing to stabilize NEK2. This suggests targeting TRIP13 may serve as a strong therapeutic target to overcome drug resistance brought by USP7 and NEK2.
In summary, this work provides strong evidence to support the notion that NEK2, USP7 and TRIP13 are potential therapeutic targets for MM disease.
- Academic Unit
- Interdisciplinary Graduate Program in Molecular Medicine
- Record Identifier
- 9983776603602771
Metrics
391 File views/ downloads
226 Record Views