Synergy of the Purine-Scaffold HSP90 Inhibitor, PU-H71, with Doxorubicin in Non-Hodgkin’s Lymphoma Cell Lines.

For over 30 years CHOP (cyclophosphamide-doxorubicin-vincristine-prednisolone) has been the standard therapy for Diffuse Large B-cell Lymphoma (DLBCL). Although DLBCL is one of the most chemotherapy-responsive human malignancies, less than 50% of newly diagnosed patients are cured with the conventional anthracycline-based chemotherapy. Novel strategies are sought out to improve the survival of these patients. Heat Shock protein 90 (HSP90) is a molecular chaperone critical for correct conformational folding of many cellular proteins. Many of those proteins are signal-transducing regulators of cell growth, DNA damage response, and survival. Cancer cells are particularly dependent on these regulators for maintenance of the transformed phenotype. Thus, HSP90 is an attractive target for cancer therapy. HSP90 inhibitors block the ATP-binding pocket and inhibit an essential ATPase activity, altering the function of the HSP90 complex and leading to destabilization and eventual degradation of mis-folded proteins. Inhibitors of HSP90 have shown promise alone or in combination with chemotherapy or radiation in preclinical studies, and are currently undergoing PhaseI/II clinical trials. Future clinical trials on their efficacy will likely explore if any additional benefit is apparent from their combination with conventional therapies. We recently demonstrated that schedule of administration of 17DMAG following Doxorubicin (DOX) ia critical for in vitro sensitivity of DLBCL cells. We examined whether the newly developed purine-scaffold HSP90 inhibitor, PU-H71, could similarly enhance the toxicity of cells to DOX when applied 24 hours after exposure to DOX. We found that sequential addition of PU-H71 to DOX resulted in reduction of DOX IC₅₀, from 400 nM to 75 nM. To evaluate the role of schedule of drug exposure as a determinant of the overall response, we analyzed the interaction of DOX and PU-H71 on cell survival. We applied standard criteria for synergy: the median effect/Combination Index (CI) method using an equimolar (1:1) ratio of DOX:PU-H71. Addition of PU-H71 to cells pretreated with DOX for 24 hours was consistently synergistic over the entire range of drug doses, with CI = 0.75 at Fa = 0.5. Although addition of PU-H71 prior to DOX was synergistic with CI = 0.7 at Fa = 0.5, this was only evident for higher doses. Only additive-to-antagonistic effects were observed at lower drug concentrations (Fa < 0.3). These results raise the question on whether synergy between PU-H71 and DOX will be observed at the concentrations achievable clinically. Conclusions: our observations provide a rationale for further preclinical testing and stress the need to consider schedule of exposure as a critical determinant of the overall response when designing clinical trials that combine HSP90 inhibitors with DNA damaging agents.