J. N. Sanchez1,2, C. Subramanian2, K. J. Kovatch3, M. S. Cohen1,2 1University Of Michigan,Department Of Pharmacology,Ann Arbor, MI, USA 2University Of Michigan,Department Of General Surgery,Ann Arbor, MI, USA 3University Of Michigan,Department Of Otolaryngology Head And Neck Surgery,Ann Arbor, MI, USA
Introduction: The 90kDa heat shock protein (Hsp90) and its clients are key regulators of multiple signaling pathways in eukaryotic cells. Its expression levels are increased 10 fold in malignancies like melanomas compared to normal tissue. Melanoma treatment has shifted to immunotherapies or targeted therapy with inhibitors targeting the MAP-kinase pathway. One challenge is that resistance to therapy especially with BRAF inhibitors occurs in a majority of patients. Since Hsp90 functions to facilitate the activation of several kinases involved in BRAF inhibitor resistance pathways, there is a high rationale that inhibition of Hsp90 would simultaneously inhibit multiple resistance pathways leading to antitumor efficacy. We have developed novel Hsp90 inhibitors that target the carboxy-terminus of the chaperone and do NOT induce the heat shock response. This response leads to dose-limiting toxicities plaguing all other Hsp90 inhibitors tested in clinical trials. We hypothesize that C-terminal inhibition of Hsp90 will simultaneously inhibit resistance pathways to rescue drug resistant melanoma cell lines.
Methods: Vemurafenib resistance was induced in validated human melanoma cell lines. Control (Mel 11) and vemurafenib-resistant (Mel 11 VeR) cell lines were treated with a Hsp90 inhibitor (KU757) or BRAF inhibitor (vemurafenib), at range of concentrations for 24hrs. Following 24hr treatment with either inhibitor, induction of apoptosis was characterized by using a caspase 3/7 luciferase reporter assay and further confirmed using flow cytometry. Flow cytometry was used for cell cycle analysis. Additionally, key resistance pathway and heat shock proteins as well as cell cycle modulators were evaluated by Western blots.
Results: Cell viability was tested in Mel 11 and Mel 11 VeR. In both cell lines, KU757 treated cells showed a similar dose-dependent inhibition of proliferation irrespective of BRAF resistance with an IC50 level of 1μ M. Next the reporter assay found that there was an equal 15 fold increase in caspase 3/7 activity for both cell lines. Further analysis of apoptosis by flow cytometry inidicated cell death by necrosis in addition to increase in caspase 3/7 activity. Analysis of cell cycle modulation using flow cytometry showed that in both cell lines, the percent of cells in G0/G1 decreased to 2.6% and 11% in Mel 11 and Mel 11 VeR respectively. Finally, Western blot analysis showed a 5 fold decrease in Akt and Erk in both cell lines with treatment compared to no treatment (p<0.01), as well as a 2 fold increase in Raf1 in vemurafenib treated cells. There was no compensatory heat shock response shown KU757 as indicated by no modulation Hsp70, Hsp32, or HSF1.
Conclusion: KU757 is equally effective in both normal and resistant melanoma cell lines inducing apoptosis, inhibiting cell growth and knocking down BRAF-inhibitor resistance pathways without inducing the heat shock response. Future translational validation in vivo will better define its clinical applications.