INHIBITION OF USP7 INDUCES SELECTIVE CANCER CELL DEATH IN CHRONIC LYMPHOCYTIC LEUKEMIA THROUGH PTEN AND INDEPENDENTLY FROM P53 STATUS
(Abstract release date: 05/19/16)
EHA Library. Carrà G. 06/09/16; 132573; E1024
Ms. Giovanna Carrà
Contributions
Contributions
Abstract
Abstract: E1024
Type: Eposter Presentation
Background
Standard immune-chemotherapy allows to achieve 60-70% response rate in CLL patients, but complete remission remains uncommon. Unsatisfactory therapeutic options still exist for higher risk groups, and in particular those harboring TP53 mutations or deletion.
Aims
We propose USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL.
Methods
Primary CLL cells were enriched in CD19+ fraction using the Milteny anti-CD19 kit and were used to investigate USP7 levels. P5091 (USP7 inhibitor) efficacy was tested in CLL cell lines and patients. Proliferation and apoptosis were evaluated respectively with CTG technology and Annexin V-FITC/Propidium PE detection by flow cytometry. The specificity of the USP7 inhibitor was verified with a pool of 5 different siRNAs able to efficiently silence USP7.
Results
Our data show that the de-ubiquitinase USP7 is aberrantly expressed in CLL. In particular, USP7 is over-expressed in about 70% of CLL CD19+ lymphocytes, both at the mRNA and protein levels. We also analyzed USP7 expression levels in an expansion cohort of a publicly available CLL patients (n=217) and 12 normal samples, where USP7 was over-expressed in CLL when compared to normal samples (****p<0.0001). We proved that USP7 is regulated at post-transcriptional level by miR-338-3p and functionally activated by Casein Kinase 2 (CK2) through phosphorylation at serine 18 residue. USP7 inhibition by P5091 as well as by specific siRNA, induces cell growth arrest and apoptosis mediated by the restoration of the nuclear pool of PTEN. Notably in primary CLL cells, P5091 treatment strongly promoted apoptosis. Moreover, USP7 inhibitor treatment of primary CLL was associated with increases ubiquitination of endogenous PTEN and consequently PTEN relocalization into the nucleus. Strikingly, TP53 deleted CLL samples and cells lines were equally subject to P5091 apoptosis induction, confirming that the USP7 inhibitor acts in a p53 independent manner. These data showed potent activity of P5091 against primary CLL.
Conclusion
We demonstrate the efficacy of a small molecule inhibitor of USP7, P5091, in vitro in cell lines and ex vivo in primary CLL samples in a P53-independent manner. Our preclinical study therefore, supports the evaluation of USP7 inhibitor as a potential CLL therapy.
Session topic: E-poster
Type: Eposter Presentation
Background
Standard immune-chemotherapy allows to achieve 60-70% response rate in CLL patients, but complete remission remains uncommon. Unsatisfactory therapeutic options still exist for higher risk groups, and in particular those harboring TP53 mutations or deletion.
Aims
We propose USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL.
Methods
Primary CLL cells were enriched in CD19+ fraction using the Milteny anti-CD19 kit and were used to investigate USP7 levels. P5091 (USP7 inhibitor) efficacy was tested in CLL cell lines and patients. Proliferation and apoptosis were evaluated respectively with CTG technology and Annexin V-FITC/Propidium PE detection by flow cytometry. The specificity of the USP7 inhibitor was verified with a pool of 5 different siRNAs able to efficiently silence USP7.
Results
Our data show that the de-ubiquitinase USP7 is aberrantly expressed in CLL. In particular, USP7 is over-expressed in about 70% of CLL CD19+ lymphocytes, both at the mRNA and protein levels. We also analyzed USP7 expression levels in an expansion cohort of a publicly available CLL patients (n=217) and 12 normal samples, where USP7 was over-expressed in CLL when compared to normal samples (****p<0.0001). We proved that USP7 is regulated at post-transcriptional level by miR-338-3p and functionally activated by Casein Kinase 2 (CK2) through phosphorylation at serine 18 residue. USP7 inhibition by P5091 as well as by specific siRNA, induces cell growth arrest and apoptosis mediated by the restoration of the nuclear pool of PTEN. Notably in primary CLL cells, P5091 treatment strongly promoted apoptosis. Moreover, USP7 inhibitor treatment of primary CLL was associated with increases ubiquitination of endogenous PTEN and consequently PTEN relocalization into the nucleus. Strikingly, TP53 deleted CLL samples and cells lines were equally subject to P5091 apoptosis induction, confirming that the USP7 inhibitor acts in a p53 independent manner. These data showed potent activity of P5091 against primary CLL.
Conclusion
We demonstrate the efficacy of a small molecule inhibitor of USP7, P5091, in vitro in cell lines and ex vivo in primary CLL samples in a P53-independent manner. Our preclinical study therefore, supports the evaluation of USP7 inhibitor as a potential CLL therapy.
Session topic: E-poster
Abstract: E1024
Type: Eposter Presentation
Background
Standard immune-chemotherapy allows to achieve 60-70% response rate in CLL patients, but complete remission remains uncommon. Unsatisfactory therapeutic options still exist for higher risk groups, and in particular those harboring TP53 mutations or deletion.
Aims
We propose USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL.
Methods
Primary CLL cells were enriched in CD19+ fraction using the Milteny anti-CD19 kit and were used to investigate USP7 levels. P5091 (USP7 inhibitor) efficacy was tested in CLL cell lines and patients. Proliferation and apoptosis were evaluated respectively with CTG technology and Annexin V-FITC/Propidium PE detection by flow cytometry. The specificity of the USP7 inhibitor was verified with a pool of 5 different siRNAs able to efficiently silence USP7.
Results
Our data show that the de-ubiquitinase USP7 is aberrantly expressed in CLL. In particular, USP7 is over-expressed in about 70% of CLL CD19+ lymphocytes, both at the mRNA and protein levels. We also analyzed USP7 expression levels in an expansion cohort of a publicly available CLL patients (n=217) and 12 normal samples, where USP7 was over-expressed in CLL when compared to normal samples (****p<0.0001). We proved that USP7 is regulated at post-transcriptional level by miR-338-3p and functionally activated by Casein Kinase 2 (CK2) through phosphorylation at serine 18 residue. USP7 inhibition by P5091 as well as by specific siRNA, induces cell growth arrest and apoptosis mediated by the restoration of the nuclear pool of PTEN. Notably in primary CLL cells, P5091 treatment strongly promoted apoptosis. Moreover, USP7 inhibitor treatment of primary CLL was associated with increases ubiquitination of endogenous PTEN and consequently PTEN relocalization into the nucleus. Strikingly, TP53 deleted CLL samples and cells lines were equally subject to P5091 apoptosis induction, confirming that the USP7 inhibitor acts in a p53 independent manner. These data showed potent activity of P5091 against primary CLL.
Conclusion
We demonstrate the efficacy of a small molecule inhibitor of USP7, P5091, in vitro in cell lines and ex vivo in primary CLL samples in a P53-independent manner. Our preclinical study therefore, supports the evaluation of USP7 inhibitor as a potential CLL therapy.
Session topic: E-poster
Type: Eposter Presentation
Background
Standard immune-chemotherapy allows to achieve 60-70% response rate in CLL patients, but complete remission remains uncommon. Unsatisfactory therapeutic options still exist for higher risk groups, and in particular those harboring TP53 mutations or deletion.
Aims
We propose USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL.
Methods
Primary CLL cells were enriched in CD19+ fraction using the Milteny anti-CD19 kit and were used to investigate USP7 levels. P5091 (USP7 inhibitor) efficacy was tested in CLL cell lines and patients. Proliferation and apoptosis were evaluated respectively with CTG technology and Annexin V-FITC/Propidium PE detection by flow cytometry. The specificity of the USP7 inhibitor was verified with a pool of 5 different siRNAs able to efficiently silence USP7.
Results
Our data show that the de-ubiquitinase USP7 is aberrantly expressed in CLL. In particular, USP7 is over-expressed in about 70% of CLL CD19+ lymphocytes, both at the mRNA and protein levels. We also analyzed USP7 expression levels in an expansion cohort of a publicly available CLL patients (n=217) and 12 normal samples, where USP7 was over-expressed in CLL when compared to normal samples (****p<0.0001). We proved that USP7 is regulated at post-transcriptional level by miR-338-3p and functionally activated by Casein Kinase 2 (CK2) through phosphorylation at serine 18 residue. USP7 inhibition by P5091 as well as by specific siRNA, induces cell growth arrest and apoptosis mediated by the restoration of the nuclear pool of PTEN. Notably in primary CLL cells, P5091 treatment strongly promoted apoptosis. Moreover, USP7 inhibitor treatment of primary CLL was associated with increases ubiquitination of endogenous PTEN and consequently PTEN relocalization into the nucleus. Strikingly, TP53 deleted CLL samples and cells lines were equally subject to P5091 apoptosis induction, confirming that the USP7 inhibitor acts in a p53 independent manner. These data showed potent activity of P5091 against primary CLL.
Conclusion
We demonstrate the efficacy of a small molecule inhibitor of USP7, P5091, in vitro in cell lines and ex vivo in primary CLL samples in a P53-independent manner. Our preclinical study therefore, supports the evaluation of USP7 inhibitor as a potential CLL therapy.
Session topic: E-poster
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