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Therapeutic Inhibition of USP7- PTEN Network in Chronic Lymphocytic Leukemia: a Strategy to Overcome TP53 Mutated/Deleted Clones

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Therapeutic Inhibition of USP7- PTEN Network in Chronic Lymphocytic Leukemia: a Strategy to Overcome TP53 Mutated/Deleted Clones Therapeutic inhibition of USP7- PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Carrà, G., C. Panuzzo, D. Torti, G. Parvis, S. Crivellaro, U. Familiari, M. Volante, et al. 2017. “Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones.” Oncotarget 8 (22): 35508-35522. doi:10.18632/oncotarget.16348. http://dx.doi.org/10.18632/ oncotarget.16348. Published Version doi:10.18632/oncotarget.16348 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33490830 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 22), pp: 35508-35522 Priority Research Paper Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/ deleted clones Giovanna Carrà1, Cristina Panuzzo1, Davide Torti1,2, Guido Parvis2,3, Sabrina Crivellaro1, Ubaldo Familiari4, Marco Volante4,5, Deborah Morena5, Marcello Francesco Lingua5, Mara Brancaccio6, Angelo Guerrasio1, Pier Paolo Pandolfi7, Giuseppe Saglio1,2,3, Riccardo Taulli5 and Alessandro Morotti1 1 Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Orbassano, Italy 2 Division of Internal Medicine - Hematology, San Luigi Gonzaga Hospital, Orbassano, Italy 3 Division of Hematology, Azienda Ospedaliera, Mauriziano, Torino, Italy 4 Division of Pathology, San Luigi Hospital, Orbassano, Italy 5 Department of Oncology, University of Turin, San Luigi Gonzaga Hospital, Orbassano, Italy 6 Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 7 Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Correspondence to: Alessandro Morotti, email: [email protected] Correspondence to: Riccardo Taulli, email: [email protected] Keywords: chronic lymphocytic leukemia, USP7, PTEN, miR181, miR338 Received: June 14, 2016 Accepted: February 20, 2017 Published: March 17, 2017 Copyright: Carrà et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disorder with either indolent or aggressive clinical course. Current treatment regiments have significantly improved the overall outcomes even if higher risk subgroups - those harboring TP53 mutations or deletions of the short arm of chromosome 17 (del17p) - remain highly challenging. In the present work, we identified USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL. We demonstrated that in primary CLL samples and in CLL cell lines USP7 is: i) over- expressed through a mechanism involving miR-338-3p and miR-181b deregulation; ii) functionally activated by Casein Kinase 2 (CK2), an upstream interactor known to be deregulated in CLL; iii) effectively targeted by the USP7 inhibitor P5091. Treatment of primary CLL samples and cell lines with P5091 induces cell growth arrest and apoptosis, through the restoration of PTEN nuclear pool, both in TP53-wild type and -null environment. Importantly, PTEN acts as the main tumor suppressive mediator along the USP7-PTEN axis in a p53 dispensable manner. In conclusion, we propose USP7 as a new druggable target in CLL. INTRODUCTION patients obtaining a complete hematological remission, while the median time to treatment failure is around Chronic lymphocytic leukemia (CLL) is a 18-20 months. Unsatisfactory therapeutic options still lymphoproliferative disorder with either an indolent exist for higher risk groups, and in particular those clinical course and long survival time or aggressive harboring TP53 mutations or deletion of the short behavior in a smaller proportion of cases [1-6]. Standard arm of chromosome 17 (del17p) [7, 8]. In addition to immuno-chemotherapy regimens achieve a 60-70% chemotherapy and anti-CD20 immunotherapy, molecular overall response rate, with only a fraction of treated insights into CLL pathogenesis and maintenance allowed www.impactjournals.com/oncotarget 35508 Oncotarget to identify novel drugs to target a variety of signaling CLL patients and representative healthy individuals, we routes to enter the clinical arena [9]. These include PI3K observed significantly increased levels of USP7 in CLL inhibitors (e.g. idelalisib) [10], BTK inhibitors (ibrutinib, samples when compared to normal cells (Figure 1B). acalabrutinib) [11, 12] and BCL2 inhibitors (venetoclax, Most CLL patients showed a USP7/GAPDH ratio higher navitoclax) [13, 14]. Del17p has been reported to be than normal CD19+ lymphocytes, indicating that USP7 present in ~7% of chemo-naïve CLL cases and 20-40% was generally over-represented in CLL (Figure 1C). The of relapsed patients and is routinely used as a prognostic biological features of enrolled patients were reported in marker, representing the sub-population facing an unmet Supplementary Table 1. USP7 is expressed both in the clinical need. Furthermore, idelalisib (Zydelig) [15] and nucleus and in the cytoplasm of representative primary ibrutinib (Imbruvica) [16] have been FDA-licensed for CLL samples and CLL cell lines, MEC-1 and EHEB CLL harboring del17p following optimistic results in (Figure 1D), as observed in other cellular models [31-34]. randomized controlled trials. Some of these drugs are Immunohistochemical analysis showed a strong positivity directed to PI3K pathway suggesting its essential role for for USP7 in 3 out of 5 CLL samples when compared to the maintenance of CLL malignant phenotype irrespective normal lymphocytes in normal bone marrow specimens of TP53 status. (Figure 1E). Finally, we analyzed USP7 expression levels Recently, Chauhan et al. showed that in a publicly available larger cohort of CLL patients (n pharmacological inhibition of the de-ubiquitinase USP7 = 217) and 12 normal samples [35]. Also in this case, strongly induces apoptosis in multiple myeloma cells USP7 was over-expressed in CLL when compared to resistant to conventional and bortezomib-based therapies normal samples (Figure 1F). Although this cohort included [17]. USP7 promotes the de-ubiquitination of various only patients with stage A of the Binet classification (i.e. targets with consequent changes in their protein levels limited-stage disease), USP7 overexpression in CLL is and cellular compartmentalization and behaves as a central highly significant and therefore these data suggest that rheostat in the definition of cell survival. USP7 plays an its overexpression may represent a common feature even essential role in tumorigenesis through the inactivation at the early stages of the disease. Altogether these data of three major tumor suppressors: p53 [18], PTEN [19, provide a rationale to investigate USP7 as a target in CLL. 20] and FoxO [21]. By affecting either their cellular compartmentalization or adjusting their protein levels, USP7 is regulated at post-transcriptional and USP7 influences the cellular fate, balancing survival post-translational levels and cell death. Both p53 [22] and PTEN [23-28] are known to play important tumor suppressive roles in CLL pathogenesis. In particular, while TP53 is in most cases Prior to investigate USP7 as a potential therapeutic genetically inactivated through point mutations or deletion target in CLL, we sought to assess the mechanisms of and correlates with resistance to standard treatments and USP7 overexpression and activation in CLL. Micro-RNAs poor prognosis, PTEN has been shown to be functionally (miRNAs) have been reported as functional players in CLL inactivated through tail-phosphorylation by the CLL pathogenesis with prognostic significance [36]. Therefore, relevant Protein Kinase 2 (CK2) [26, 27, 29, 30]. Here we performed a bioinformatic survey of publicly available we investigate the mechanisms of USP7 regulation in datasets [35] returning a list of miRNAs potentially able CLL, explore the functional role of USP7 in the broader to target the USP7 3′-UTR (Supplementary Figure S1A). context of its signaling partners (i.e. the USP7-PTEN The calculated Pearson correlation coefficient was highly network) and provide evidences supporting its potential significant for an inverse correlation between USP7 and therapeutic exploitation. Finally, we discuss the ability of miR-338-3p and miR-181b (Supplementary Figure S1B USP7 inhibitor to effectively target CLL cells regardless and S1C). Thereby, we firstly subcloned the miR-338- of their TP53 status. 3p responsive element (MRE) of USP7 (Supplementary Figure S1D, upper panel) downstream to a luciferase RESULTS construct and a reporter assay showed that miR-338-3p could directly down-regulate USP7 at post-transcriptional level (Supplementary Figure S1D, lower panel). Similar data were obtained with miR-181b response element USP7 is strongly up-regulated in CLL samples (Supplementary Figure S1E upper
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