P204
ROLE OF HAUSP/PTEN NETWORK IN CHRONIC LYMPHOID LEUKEMIA PATHOGENESIS AND THERAPY
G. Carra1,* C. Panuzzo1, D. Torti1, G. Parvis1, S. Crivellaro1, A. Guerrasio1, G. Saglio1, A. Morotti1
1Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, Turin, Italy
Background: PTEN tumor suppressor was shown to play a role in Chronic Lymphocytic Leukemia (CLL). In particular, it was demonstrated that PTEN is phosphorylated in the tail by Casein Kinase II, favoring its inactivation. Further more, it was also proposed that PTEN levels are significantly reduced in some CLL patients. PTEN tumor suppressive functions are also regulated by proper cellular compartmentalization through mono-ubiquitination. In particular, PTEN mono-ubiquitination promotes PTEN nuclear localization, while PTEN de-ubiquitination by HAUSP promotes PTEN nuclear exclusion with dramatic consequences
on the tumor suppressive function.
Aims: Assessment of the biological role of CKII/HAUSP/PTEN network in CLL in order to evaluate HAUSP as a rheostat in the regulation of PTEN. Evaluation of the therapeutic role of HAUSP inhibitors in CLL.
Methods: Primary CLL cells were collected from untreated and informed patients accordingly to ethical committee approved protocol, during routinely diagnostic procedures. Cells were enriched in CD19 fraction using the Miltenyi anti CD19 kit. CLL CD19 cells were used for different analyses. Immunofluorescence was performed to investigate PTEN cellular compartmentalization.
Protein samples were used for HAUSP and PTEN protein levels quantification and evaluation of those proteins regulated by HAUSP (p53, p21, MDM2). The same material was used for PTEN ubiquitination, PTEN phosphatase assay and Casein Kinase II kinase assay. Transfected CLL cell lines with GFP-PTEN and mutant constructs were used to evaluate the contribution of PTEN in promoting growth arrest and/or apoptosis induction. HAUSP inhibitors were used
in CLL cell lines and proliferation and apoptosis were evaluated with MTT technology and Annexin V-FITC/Propidium PE detection by flow cytometry.
Results: By immunofluorescence we observed that a portion of CLL patients is characterized by PTEN nuclear exclusion. PTEN protein levels did not correlate with cellular compartmentalization, suggesting that the two phenomena
are regulated by different mechanisms. Forcing PTEN expression into the nucleus of a CLL cell line, with a PTEN-NLS expressing vector, was associated with strong apoptosis induction and growth arrest. These observations suggest that PTEN is functionally inactivated in CLL. Next, we assessed PTEN ubiquitination in CLL primary cells. We observed that PTEN is de-ubiquitinated, clearly
demonstrating a direct role of HAUSP in PTEN nuclear exclusion of CLL cells. Our data suggested a dual regulatory mechanisms that activate HAUSP towards PTEN. HAUSP could be either over-expressed and/or regulated by Casein Kinase II, which phosphorylates HAUSP on serine residues. Interestingly, treatment of CLL cells with HAUSP small molecule inhibitors is associated with re-localization of PTEN into the nucleus, which in turn promotes cell growth arrest and apoptosis induction.
Summary and Conclusions: Our data demonstrate that (1) PTEN is functionally inactivated by nuclear exclusion in CLL; (2) PTEN can be reactivated
in CLL through HAUSP and Casein Kinase II inhibitors, with significant therapeutic implications.
