Abstract B105: HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes
Authors: Mbofung Rina M. , McKenzie Jodi A. , Malu Shruti , Liu Chengwen , Peng Weiyi , Kuiatse Isere , Williams Leila , Devi Seram , Wang Zhe , Tieu Trang , Heffernan Tim , Davis Richard E. , Amaria Rodabe , Hwu Patrick 
Published: November 2016

Abstract
Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired patient derived human melanoma tumor samples and TILs were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst the top compounds that improved T cell mediated cytotoxicity of treated tumor cells. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of human cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P &lt; 0.0001). Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon response genes including IFIT1, IFIT2, IFIT3. We confirmed these findings with quantitative real time PCR and western blot analyses and found IFIT1, IFIT2 and IFIT3 to be consistently upregulated across multiple melanoma cell lines following treatment with ganetespib. We next sought to verify the importance of the IFIT genes in the synergy observed between ganetespib treatment and T cell killing. First, we overexpressed IFIT1, IFIT2 and IFIT3 in human melanoma cell lines to recapitulate the improved sensitivity of the human melanoma cell lines to T cell killing following treatment with ganetespib. We then co-cultured these cells with their autologous T cells and found that overexpressing IFIT1, IFIT2 and IFIT3 mimicked the effects of ganetespib by increasing the sensitivity to T cell killing over the GFP control. On the other hand, silencing IFIT1, IFIT2 and IFIT3 simultaneously, abrogated the synergy between ganetespib and T cell killing. We are further elucidating the role of these genes in lowering the apoptotic threshold of cancer cells and contributing to the synergy of ganetespib and immunotherapy. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses.
Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Weiyi Peng, Isere Kuiatse, Leila Williams, Seram Devi, Zhe Wang, Trang Tieu, Tim Heffernan, Richard E. Davis, Rodabe Amaria, Patrick Hwu. HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B105.