Author ORCID Identifier

orcid.org/0000-0003-3872-3143

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Immunology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Patrick Hwu, M.D.

Committee Member

Willem Overwijk, Ph.D.

Committee Member

Richard Eric Davis, M.D.

Committee Member

Giulio Draetta, M.D., Ph.D.

Committee Member

Brendan Lee, M.D., Ph.D.

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 tumour infiltrating T cells (TILs), conferred a clinical response rate of ~50%. Treatment with anti-CTLA4 and anti –PD1 therapy, conferred response rates of up to 50%, greatly improving the overall survival of patients with advanced melanoma amongst other cancer types. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumour shrinkage can be appreciated. Thus, understanding what tumour intrinsic pathways contribute to non-responsiveness to immunotherapies and their effect on T cells, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of cancer patients.

In order to identify possible tumour intrinsic pathways that could be perturbed to improve responses 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 tumour cells to T cell mediated killing. Paired tumour samples and TILs from melanoma patients 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. Heat shock protein 90 (HSP90) inhibitors amongst compounds that improved T cell mediated cytotoxicity. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of both human and murine cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumour model show that ganestespib in combination with immune checkpoint blockade, resulted in superior antitumour effect and survival compared to either treatment alone. Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon alpha (IFN-α) response genes including IFIT1, IFIT2 and IFIT3. Silencing IFIT genes abrogated the synergy observed with ganetespib treatment and T cell mediated killing, suggesting that the IFN-α response pathway plays an important role in this combination therapy. This work highlights the importance of IFIT genes in response to T cell based immunotherapy and 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.

To understand what factors influence CD8 T cell effector function at the tumour site, we used a murine adoptive cellular therapy model in which B16, a gp100-expressing tumour cell line was implanted in mice and treated with transgenic Pmel T cells, which recognize gp100 in the context of H-2Db.Transferred Pmel T cells were recovered from the spleen and tumour of the mice. To interrogate novel pathways which may inhibit the functions of tumour-reactive T cells at the tumour site, microarray and genome-wide gene expression analyses, were used to characterize the differential expression profiles among Pmel T cells from different groups. 720 genes were differentially expressed by T cells recovered from the tumour site, when compared with those recovered from the spleen. Amongst them, was the transcription factors Runx2, a gene whose role has not been described in T cells, to be one of the genes that were differentially expressed. In addition, we found that when Runx2 was absent in T cells they did a better job at controlling tumour growth and vice versa. Further investigation revealed that Runx2 regulated processes in T cells such as cytokine production and differentiation status, processes which are important in controlling tumour growth. We are currently focused on understanding how Runx2 controls these processes in an effort to provide ways to improve existing immunotherapies or discover new ones.

Keywords

Immunotherapy, HSP90, Interferon, Runx2, T cells, IFIT, Combination therapy, Memory T cells, Terminal effector T cells

Available for download on Tuesday, October 31, 2017

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