Presentation Title

Evaluating the Efficacy of Cancer Immunotherapy in Mouse Melanoma by Manipulating IFN-ɣ Signaling

Faculty Mentor

Ke Shuai

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 3:15 PM

Location

BSC-Ursa Minor 96

Session

Poster 3

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Current cancer treatment improvement has shifted towards immunotherapy. Therapy blocking immune checkpoint inhibitors, such as PD1/PD-L1, shows promising results in some melanoma patients, while the majority of patients do not respond. We are using two mouse melanoma cell lines, B16 F10 and YUMM 2.1, to explore the mechanism of resistance to immune therapy. B16 F10 has limited response to PD1/PD-L1 immunotherapy, whereas YUMM 2.1 is a strong responder. IFN-γ activates the JAK/STAT pathway, which turns on the genes necessary for immunotherapy response. The Protein Inhibitor of Activated STAT (PIAS) proteins are known STAT signaling inhibitors that block IFN-γ signaling. We hypothesize that manipulating PIAS1 will alter immunotherapy sensitivity. We asked if IFN-γ treatment can induce PIAS1 protein levels. Our Western blot showed greater PIAS1 induction in B16 F10 compared to YUMM 2.1. We also found differences in expression of IFN-γ-induced genes between the cell lines. We performed PIAS1 shRNA knockdowns to manipulate protein expression. PIAS1 knockdown single colonies were generated and verified through Western blot. With PIAS1 knockdown cells, we tested gene expression changes by treating these cells with IFN-γ and analyzing qPCR data for IFN-γ-induced genes. We hypothesize that the removal of PIAS proteins will confer greater sensitivity to PD1/PD-L1 immunotherapy due to the enhanced IFN-γ signal transduction. By manipulating negative regulators, we hope to translate these murine data into human melanoma cases and develop promising therapeutics.

Summary of research results to be presented

My data will consist of Western blots images as well as analyses of qPCR data. For the Western blots, I will demonstrate that IFN-γ treatment induces a change in PIAS1 expression in mouse melanoma cells. I conducted TNF-α treatment on my cells and ran a Western blot to analyze PIAS1 induction in the cytoplasm and nucleus. This Western blot shows that PIAS1 activation through phosphorylation is important in the nucleus. I will also demonstrate with a Western blot that I have successfully knocked down PIAS1 in B16 F10 and YUMM 2.1 cells. For my qPCR data, I will show changes in certain IFN-γ-induced genes, such as chemokines CXCL9 and CXCL10 and other downstream genes, that occur from IFN-γ treatment, and I will also compare the treatment results between parental B16 F10 cells, B16 F10 transfect control cells, and B16 F10 PIAS1 knockdown cells.

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Nov 18th, 2:15 PM Nov 18th, 3:15 PM

Evaluating the Efficacy of Cancer Immunotherapy in Mouse Melanoma by Manipulating IFN-ɣ Signaling

BSC-Ursa Minor 96

Current cancer treatment improvement has shifted towards immunotherapy. Therapy blocking immune checkpoint inhibitors, such as PD1/PD-L1, shows promising results in some melanoma patients, while the majority of patients do not respond. We are using two mouse melanoma cell lines, B16 F10 and YUMM 2.1, to explore the mechanism of resistance to immune therapy. B16 F10 has limited response to PD1/PD-L1 immunotherapy, whereas YUMM 2.1 is a strong responder. IFN-γ activates the JAK/STAT pathway, which turns on the genes necessary for immunotherapy response. The Protein Inhibitor of Activated STAT (PIAS) proteins are known STAT signaling inhibitors that block IFN-γ signaling. We hypothesize that manipulating PIAS1 will alter immunotherapy sensitivity. We asked if IFN-γ treatment can induce PIAS1 protein levels. Our Western blot showed greater PIAS1 induction in B16 F10 compared to YUMM 2.1. We also found differences in expression of IFN-γ-induced genes between the cell lines. We performed PIAS1 shRNA knockdowns to manipulate protein expression. PIAS1 knockdown single colonies were generated and verified through Western blot. With PIAS1 knockdown cells, we tested gene expression changes by treating these cells with IFN-γ and analyzing qPCR data for IFN-γ-induced genes. We hypothesize that the removal of PIAS proteins will confer greater sensitivity to PD1/PD-L1 immunotherapy due to the enhanced IFN-γ signal transduction. By manipulating negative regulators, we hope to translate these murine data into human melanoma cases and develop promising therapeutics.