Functional Characterization of NAD Dependent De-Acetylases SIRT1 and SIRT2 in B-Cell Chronic Lymphocytic Leukemia (CLL)

Introduction: CLL is characterized by abnormal proliferation and accumulation of mature CD5 positive B-lymphocytes in blood, bone marrow, spleen and lymph nodes.  Outcome and survival is determined in part by the presence of 11q deletions and17p deletion/TP53 mutation with complex karyotype. Sirtuins are NAD+ dependent ADP-ribosyl transferases with evolutionary conserved function in cellular metabolism and chromatin regulation. Seven sirtuins (SIRT1-SIRT7) have been identified in mammals at distinct subcellular locations and targeting different substrates. SIRT1, 2, 6, and 7 are primarily found in the nucleus, SIRT2 in the cytoplasm and SIRT3, 4, 5 in the mitochondria. Sirtuins are associated with cancer as they deacetylate cancer associated transcription factors, and SIRT1 is overexpressed in acute myeloid leukemia, colon and prostate cancers. Several studies reported SIRT2 as a tumor suppressor as it is down regulated in human gliomas. SIRT1 and SIRT6 are reported to be significantly increased in CLL. We hypothesized that sirtuins play an important role in the development and maintenance of CLL and might therefore be a target.

Methods:   We measured SIRT 1 and 2 expression in fresh primary CLL cells, in the B-cell pro-lymphocytic cell lines JVM-3 and MEC-2 and by data-mining of the Oncomine microarray gene expression datasets. Oncomine is a bioinformatics initiative that collects, standardizes, analyzes, and delivers cancer transcriptome data to the biomedical research community.  We then inhibited SIRT activity in primary CLL cells and cell lines by pharmacologic inhibitors EX-529 and sirtinol, and by knock down using shRNA in cell lines and then measured cell viability, apoptosis, reactive oxygen species formation and mitochondrial membrane potential. To determine the metabolic contribution to SIRT activity, we studied the effect of SIRT inhibition under conditions of nutrient deprivation.

Results: We observed an increase in SIRT1 mRNA expression in CLL by data mining of the independent microarray dataset in the Oncomine database, with a total of 2022 leukemia samples and 74 normal controls (Figure1).  SIRT1 was significantly up regulated in CLL compared with normal PBMC as well as other leukemia types.  We found that SIRT inhibitors EX-527 and sirtinol impair cell growth (IC50 50-100 microM for EX-527 and 10-20 microM for sirtinol), cause apoptosis (>2-fold increase in apoptosis in cell lines JVM-3 and MEC-2), induce ROS production (up to 90% increase in mean fluorescence intensity (MFI) with EX-527 and sirtinol), loss of mitochondrial membrane potential, (MFI from 4 to <1 after treatment with SIRT inhibitors) and increase alpha-tubulin acetylation in primary CLL cells and cell lines. Using shRNA knock down of SIRT1 and SIRT2 in JVM-3 and MEC-2 cell lines, we showed that expression of both proteins is crucial for the survival of these cells. Furthermore, studies in nutrient deprived conditions suggest a role of SIRT in metabolism in CLL.

Conclusion:  These findings suggest that CLL cells are characterized by increased expression and function of SIRT1 and SIRT2, both directly inhibited by SIRT inhibitors. SIRT1 and SIRT2 inhibition using specific inhibitors could be a novel therapeutic approach for the treatment of CLL and other SIRT expressing hematologic malignancies.

Figure 1