Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones

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.

Keywords: PTEN; USP7; chronic lymphocytic leukemia; miR181; miR338.

Figure 1. USP7 is strongly up-regulated in CLL samples

A. Quantification of mRNA levels in 5 normal CD19⁺ lymphocytes and 19 CLL samples. *p < 0.05. B. Primary CD19⁺ lymphocytes from two representative normal individuals and ten CLL patients were analyzed for USP7 protein expression. C. Quantification of USP7/GAPDH ratio in 5 normal CD19⁺ lymphocytes and 19 CLL samples. **p < 0.01. D. Western Immunoblot of cytoplasm/nuclear fractions in CLL cell lines model and two representative primary CLL samples. E. USP7 immunohistochemical of human biopsies in one representative normal bone marrow and two CLL specimens. F. Box-plot of USP7 mRNA levels in normal lymphocytes (n = 12) compared to CLL primary cells (n = 217). ****p < 0.0001.

Figure 2. USP7 Post-translational modifications in CLL

A. Representation of USP7 isoforms segments highlighting the only isoform containing serine-18. B. Quantification of USP7 isoform 1 (Iso_1) and isoform 3 (Iso_3) mRNA levels in CD19⁺ lymphocytes from 19 CLL samples. **p < 0.01. C. Quantification of USP7 isoform 1 and 3 mRNA levels in MEC-1 cells. **p < 0.01. D. Kinase assay with purified USP7 protein and immunoprecipitated CK2. The assay was performed in the absence or presence of 60 μM TBB. p-SER: phospho-serine. E. Upper panel: Purified tumor cells from normal CD19⁺ and CLL patients were analyzed for the indicated proteins. Lower panel: quantification of pSer18 USP7 expression level, normalized on total USP7. **p < 0.01.

Figure 3. Targeting USP7 induces growth inhibition and apoptosis in CLL cell lines

A. Proliferation analysis of MEC-1 cells treated with USP7 inhibitor (P5091) for the indicated times and concentrations. The number of cells at day 0 was set at 1 unit. Insert: lysates from control and P5091-treated MEC-1 cells were analyzed for p21 expression level. B. Cell cycle distribution of MEC-1 cells treated or not with P5091 for 3 days. C. Quantification and representative images of soft-agar growth assay in MEC-1 cells treated with the indicated concentrations of P5091 for 15 days. ***p < 0.001. D. Upper panel: evaluation of apoptosis in MEC-1 cells treated with the indicated concentrations of P5091 for 24 hrs. ***p < 0.001. Lower panel: cleaved-caspase-3 Western Immunoblot in the same conditions. E. Proliferation analysis in MEC-1 cells infected either with control (siCTR) or USP7-directed (siUSP7) siRNAs (insert). *p < 0.05; **p < 0.01. F. Apoptosis analysis of MEC-1 cells described in E. *p < 0.05.

Figure 4. USP7 inhibition affects PTEN delocalization

A. PTEN-ubiquitination assay in MEC-1 cells treated with 16 μM P5091 for 2 and 4 hours. B. USP7 Western Immunoblot upon cytosol/nuclear fractionation of MEC-1 cells treated with P5091 at a concentration of 16 μM for 2 and 4 hours. C. Immunofluorescence assay showing PTEN cellular compartmentalization in MEC-1 cells treated with 4.2 μM P5091 for 24 hours. Anti-PTEN antibody (green); Propidium iodide (red). D. Proliferation analysis of MEC-1 cells expressing GFP-PTEN-WT and GFP-PTEN-NLS. Cell growth was quantified by counting cells with trypan blue exclusion of dead cells at each time point. ***p < 0.001. E. Percentage of apoptosis in GFP-PTEN transfected MEC-1 cells. *p < 0.05.

Figure 5. P5091 affects USP7/PTEN network also in primary CLL samples

A. Left panel: Representative immunofluorescence images on CD19⁺ lymphocytes from peripheral blood of normal and CLL individuals, stained with anti-PTEN antibody (green) and Propidium iodide (red). Right panel: Western Immunoblot of cytoplasm/nuclear fractions in two representative primary CLL samples. B. Quantification of PTEN cellular compartmentalization in CD19⁺ lymphocytes from 5 normal and 14 CLL samples. Data represent mean ±SD of > 50 cells analyzed from each sample and expressed as percentage. C. Apoptosis analysis of CD19⁺ lymphocytes from normal (n = 3) and CLL (n = 9) individuals treated with P5091 for 24 hours. Percentage of each sample was normalized to corresponding untreated condition. ***p < 0.001. D. PTEN ubiquitylation assay in CLL primary cells treated with DMSO or P5091 (16 μM) for 2 hours. E. PTEN cellular compartmentalization (green) in a representative PTEN nuclear-excluded CLL sample before and after treatment with P5091 (4.2 μM) for 24 hours. F. Model of USP7 network in CLL.