TRIS (DIBENZYLIDENEACETONE) Dipalladium a Small-Molecule Palladium Complex Is Effective in the Induction of Apoptosis for B-Chronic Lymphocytic Leukemia B-Cells

In an attempt to better treat B-Chronic Lymphocytic Leukemia (CLL) patients, we have examined a unique organometallic complex called Tris (dibenzylideneacetone) dipalladium (Tris DBA). This novel agent is a small-molecule palladium complex shown to have antiproliferative activity against melanoma cells and possible antitumor activity. Tris DBA is known to inhibit activation of signaling molecules such as PI3K and STAT-3, critical factors in CLL B-cell survival and resistance to therapeutic agents. Because B-CLL remains incurable and treatment strategies challenging, our findings may be useful in pursing new therapies to improve patient care. Therefore, this information prompted us to investigate whether Tris DBA could induce apoptosis in CLL B-cells.

Method of Approach: Primary leukemic B-cells from CLL patients were cultured in serum free media and treated at 2 × 10⁶ cells per ml with vehicle or various doses of Tris DBA (1 – 10 μg/ml) for 24 – 72 hours. After treatment, cells were analyzed for apoptosis by a FITC-labeled annexin-V/propidium iodide assay using the FACScan. To assess changes in anti-apoptotic proteins, CLL-B cells were cultured for 24 hours with either vehicle or 2.5μg/mL of Tris DBA. Total protein extracts were used for immunoblot methodology with specific antibodies. In some experiments we developed primary marrow stromal cells (MSC) that when nearly confluent were used for co-culture experiments in the presence of freshly isolated CLL B-cells with various chemotherapeutic drugs and/or Tris DBA. Finally, we compared CLL patients with known immunoglobulin heavy chain variable region (IgVH) status because of the clear differences in clinical outcome for these two prognostic subgroups.

CLL B-cells were shown to be sensitive to Tris-DBA with a steep dose response curve that significantly increased apoptosis over control levels as early as 24 hours of culture. Thus the LD50 of Tris DBA was between 2.5 and 5.0 μg/ml for the CLL B-cells who were IgVH mutated while between 5 and 7.5μg/ml for the unmutated status CLL B cells (n=10). Normal B or T lymphocytes were found to be equally sensitive to Tris DBA when compared to CLL B-cells however mesenchymal stromal cells (MSC) from CLL marrow (CLL MSC) were far less sensitive to the Tris DBA drug. Thus the LD50 for normal T cells, B cells or NK cells was between 2.5 and 5.0 μg/ml while the LD50 for the MSC was not reached over the dose range of 1–10 μg/ml. When CLL B-cells were added to CLL derived primary MSC in the presence of increasing concentrations of Tris DBA (2.5–10 μg/ml), the cytotoxic impact of Tris DBA on CLL B-cells could be overcome in the presence of MSC. To delineate the mechanism of apoptosis induction of CLL B-cells, we treated CLL B-cells with sub-lethal dose of Tris DBA and prepared cell lysates for Western blot analyses. We found that the anti-apoptotic proteins XIAP and Bcl-2, but not Mcl-1, were down regulated in Tris- DBA-treated leukemic B-cells. In addition, phosphorylation levels of Akt and STAT-3 were inhibited with exposure to Tris DBA as measured by Western blot using specific antibodies. Finally, we studied if Tris DBA could be synergistic or additive with fludarabine in induction of apoptosis of CLL B- cells. Interestingly, we found that in 2 of 3 assays there was synergy between Tris DBA and fludarabine in the enhancement of CLL B-cell apoptosis.

We have established that a unique compound, Tris dipalladium, is effective at inducing apoptosis in CLL B-cells whether these leukemic cells are IGVH mutated (low risk) or unmutated (high risk). In addition this compound can modify the levels of XIAP and Bcl-2, Akt and STAT3 activation in the leukemic B-cells. Importantly, by itself the Tris DBA is effective even in the presence of stromal cells albeit losing some effectiveness at high doses in the co-culture and can generate synergy when added to a purine nucleoside for induction of B-cell apoptosis. In total these findings support future clinical applications of this compound in treating CLL patients.

Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees. Arbiser:Natuderm: Patents & Royalties.