Targeting The Unfolded Protein Response As A Therapeutic Approach In Hematological Malignancies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The unfolded protein response (UPR) is a complex signaling pathway activated in response to endoplasmic reticulum stress. In recent years, the UPR has been implicated in cancer and chemosensitivity, particularly solid tumors. We have investigated the potential value of targeting the UPR as a novel therapeutic approach in hematological malignancies using a panel of cell lines representing AML, lymphoma and multiple myeloma. UPR activation was determined from expression of the key ER chaperones GRP78 and GRP94 and UPR mediators using Western blot analysis. The effect of cytotoxic agents and agents that induce (thapsigargin, tunicamycin), target (versipelostatin) or silence (siRNA) UPR proteins were studied, alone and in combination, using cytotoxicity, apoptosis and colony formation assays. The UPR was constitutively active in these hematological cancer cell lines, with differential activation of key UPR proteins across the tumor types studied. There were also differences in UPR activation between the hematological cell line panel, peripheral blood mononuclear cells and the colorectal cancer cell line HT-29. Further UPR activation was seen at the translational level following minimally toxic (EC5), and cytotoxic (EC25 and EC50), concentrations of ER stress inducers, proteasome and HSP90 inhibitors. A number of strategies were used to study the effect of UPR modulation on chemosensitivity. Minimally toxic concentrations of the ER stress inducer thapsigargin protected cells from cytotoxic agents, with a reduction in antiproliferative drug effect. The activity of the novel small molecule versipelostatin, reported to downregulate the ER molecular chaperones GRP78 and GRP94, was also investigated. The downregulation of these chaperone proteins previously reported in solid tumor cell lines (Park et al. 2004) was confirmed in HT-29 cells, but downregulation was not observed in the hematological cell lines studied, although versipelostatin was an effective cytotoxic agent at low micromolar concentration. Combination experiments with the chemical chaperone 4-phenylbutyric acid (PBA) resulted in a small increase in apoptosis when PBA was combined with ER stress inducing agents. However, PBA also showed HDAC inhibitory activity at the concentrations used, suggesting the interaction seen may be due to HDAC inhibition in addition to, or instead of, its chemical chaperone activity. Finally, siRNA mediated silencing of GRP78 and GRP94 in the THP1 (AML) and U266 (multiple myeloma) cells resulted in a decrease in the targeted protein, but showed only minimal effects on the activity of chemotherapy. In conclusion, the UPR is activated in these hematological cancer cell lines and plays a complex role in chemosensitivity. In contrast to previous reports in solid tumor cells, modulating the UPR in these hematological malignancies had only a modest effect on sensitivity to cytotoxic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2793. doi:1538-7445.AM2012-2793