JVM-3

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

Sirtuins (SIRT) proteins play an important role in the survival and drug resistance of cancer cells. SIRT1 and SIRT2 protein expression were quantified in fresh CLL samples from 9 patients and 2 cell lines derived from B-PLL (JVM-3 and MEC-2) using Western blotting. As shown in Fig. 1A, both SIRT1 and SIRT2 proteins were overexpressed in all the CLL samples examined, however, SIRT2 levels were lower than SIRT1 in most samples (Fig. 1B). By contrast, there was little difference in the protein levels of SIRT1 and SIRT2 in the 2 PLL cell lines JVM-3 and MEC-2 as measured by Western blotting (Fig. 1C) followed by quantitation of relative protein expression (Fig. 1D). These results demonstrate that both SIRT1 and SIRT 2 are overexpressed in CLL patients and PLL cell lines.

The effect of SIRT inhibitors, sirtinol, and EX-527 in PBMC was tested by analyzing cell viability by WST1. EX-527 and sirtinol treatment inhibited cell growth in all the samples tested with an IC50 ranging from 50-100 µM for Ex-527 and 10-20 µM for sirtinol. Cell viability was significantly inhibited in the presence of both inhibitors in a dose-dependent manner at 24-72 hr. (Fig. 2A). Similar effects of EX-527 and sirtinol were observed on JVM-3 and MEC-2 cell lines (Fig. 2B). Both drugs induced significant apoptosis at all the concentrations tested in most of the samples (Fig. 2C) as well as in the cell lines (Fig. 2D). Taken together, these data suggest that inhibition of SIRT1 results in cellular apoptosis in CLL patient samples and relevant cell lines.

Fig. 1 SIRT1 and SIRT2 are overexpressed in CLL cell lines (JVM-3 and MEC-2). (Bhalla S and Gordon LI, 2016)

Fig. 2 Pharmacological inhibition of Sirtuins affects cell viability and induces apoptosis in CLL cell lines (JVM-3 and MEC-2). (Bhalla S and Gordon LI, 2016)

Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B-CLL

The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and primary B-chronic lymphocytic leukemia (B-CLL) patient samples. As shown in Fig. 3, the combined treatment induced a reduction of cell viability significantly higher for the single drugs in all B leukemic cell lines analyzed. The induction of apoptosis, rather than the cell cycle block mainly accounted for the synergistic anti-leukemic activity of the Ibrutinib/Nutlin-3 combination. The analysis of apoptosis showed that Ibrutinib alone induced modest levels of apoptosis in all cell lines, while the Ibrutinib/Nutlin-3 combination significantly increased the percentage of apoptosis for the treatment with the single drugs used alone, in both p53wild-type and p53mutated B leukemic cell lines (Fig. 4).

It is well known that the pathogenesis of B-CLL is characterized by alterations of cellular signaling pathways. Exposure of B leukemic cell cultures to Ibrutinib resulted in inhibition of BTK auto-phosphorylation at tyrosine 223 (caused by the inhibition of kinase activity by the binding of Ibrutinib) coupled to a significant reduction of the phosphorylation of the MAPK survival pathway modulators ERK1/2, starting at early time points post drug exposure (Fig. 5A). Moreover, by assessing phosphorylation levels through magnetic-plex assays, a rapid down-modulation of the PI3K survival pathway was observed, as documented by the reduction of both Akt and m-TOR phosphorylation, which was validated also by Western blotting (Fig. 5B). No significant modulation of the p53 pathway was observed in p53mutated B cells following any treatment (Fig. 5C). Western blotting analysis on cellular lysates from B-leukemic cell lines highlighted that the exposure to Ibrutinib was associated to up-regulation of phospho-H2A.X, responsible for the DDR-signal amplification, and that this response was further enhanced by the combination with Nutlin-3 (Fig. 5D).

Fig. 3 Evaluation of cytotoxic effect in response to Ibrutinib and Nutlin-3 used alone or in combination in B leukemic cell lines. (Voltan R, et al., 2016)

Fig. 4 Effects of Ibrutinib/Nutlin-3 combination on cell apoptosis in B leukemic cell lines. (Voltan R, et al., 2016)

Fig. 5 Intracellular pathway mediating the anti-leukemic activity of Ibrutinib/Nutlin-3 combination. (Voltan R, et al., 2016)