Anti-Apoptotic Bcl2 and Bcl-Xl Proteins Involved in Stroma-Induced Drug Tolerance to Hsp70 and Hsp90 Inhibitors in Leukemia and B-Cell Lymphoma

Multiple studies have demonstrated that the bone marrow stromal microenvironment contributes to the survival of hematologic malignant cells, eventually leading to relapse. However, molecular mechanisms associated with this stromal niche remain unclear. The human bone marrow stromal cell lines, HS-5 and HS-27, provide physical contact with hematologic cells, while HS-5 cells secrete more growth factors and cytokines than HS-27 stromal cells. Our objective is to dissect the mechanisms underlying stromal-mediated drug tolerance in leukemia and lymphoma cells, which could potentially lead to novel therapies for various leukemia.

A panel of leukemia and B-cell lymphoma cell lines were used in this project, including Kasumi1 (AML: Acute Myeloid Leukemia) and OCILy1 (DLBCL: Diffuse Large B-Cell Lymphoma) cells and their respective sub-lines resistant to heat shock protein-70 and −90 (HSP70/90) inhibitors. To determine the ability of stromal cell lines to confer tolerance to HSP-inhibitors, Kasumi1 and OCILy1 (sensitive and resistant) cells were cultured alone or in the presence of the HS-27 or HS-5 cells with HSP70 inhibitor or HSP90 inhibitor for 48h. The resulting cultures were then harvested and analyzed for apoptosis and by western blot. Both HS-5 and HS-27 stromal cells markedly protected OCILy1 and Kasumi1 cells from HSP70 inhibitor induced apoptosis. At a dose of 0.5 μM, % apoptotic cells were 74.0±1.6% for OCILy1 alone, 38.3±2.1% for OCILy1 with HS-5 and 42.2±1.8% for OCILY1 with HS-27. At a dose of 1 μM of HSP90 inhibitor, apoptosis rate are 61.9±1.5% for OCILy1 alone, 28.2±2.2% for OCILy1 with HS-5 and 36.4±1.9% for OCILy1 with HS-27. A similar HSP inhibitor induced apoptosis was also observed in Kasumi1 cells. In contrast, both Kasumi1 and OCILy1 HSP70/90 inhibitor resistant sub-lines in the presence or absence of the stromal cells did not respond to treatment with respective inhibitors. Further study reveals the stromal cells up-modulated the expression of the anti-apoptotic proteins Bcl2 and Bcl-xL in both Kasumi and OCILY1 cells.

Our results demonstrate that the stromal niche is able to mediate tolerance to HSP70 and HSP90 inhibitors in Leukemia and B-cell lymphoma via up-regulation of antiapoptotic proteins such as Bcl2 and Bcl-xL. The Bcl2 protein is deregulated and plays a crucial role in diffuse large B-cell lymphoma (DLBCL) with the t(14;18) translocation. Our finding elucidates one of the drug-specific mechanisms that suggest a promising combination therapy targeting both HSP70 and HSP90 to reduce antineoplastic resistance and relapse, and thereby improve survival for patients with leukemia and lymphoma.

No relevant conflicts of interest to declare.