HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes

Abstract

T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.Many patients fail to respond to T cell based immunotherapies. Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.

Fig. 1

HSP90 inhibition enhances T-cell mediated killing of melanoma cells. a Tableau depiction of screen results from patient-derived cell line 2549. HSP90 inhibitors 1—17-DMAG, 2—BIIB021 and 3—17-AAG are highlighted. b Cleaved caspase 3 percentage following treatment with varying concentrations of ganetespib and autologous TILs in human melanoma cell lines 2549 and 2400. c Isobolograms depicting the synergism between ganetespib and T-cell killing. Points >1 indicate antagonism, points = 1 indicate additivity and points <1 indicate synergism. An unpaired two tailed Student t-test was performed with **P < 0.01; ****P < 0.0001. Data are a representation of at least two independent studies

Fig. 2

HSP90 inhibitor effect on T-cell-mediated killing requires IFIT genes. a Gene expression analysis of cell lines treated with ganetespib. b, c Quantitative real-time PCR (qRT-PCR) and western blot analysis showing upregulation of IFIT1, IFIT2 and IFIT3 following ganetespib treatment in human melanoma cell line 2400. D = DMSO and G = ganetespib. Interferon alpha (IFN-α) used as a positive control. An increase in cleaved PARP indicates efficacy of the HSP90 inhibition by ganetespib. d qRT-PCR to verify silencing of IFIT1, IFIT2 and IFIT3 in 2400. Control = cell line transduced with scrambled shRNA and IFITall = cell line transduced simultaneously with IFIT1, IFIT2 and IFIT3 shRNAs. e 2400 Control and IFITall cell lines treated with ganetespib at 250 nM, co-cultured with autologous T cells and assayed for cleaved caspase 3 by flow cytometry. f qRT-PCR verifying overexpression of IFIT1, IFIT2 and IFIT3 over GFP control in 2400. g 2400 GFP and IFIT overexpressing cell lines co-cultured with autologous T cells and assayed for cleaved caspase 3. h Western blots showing a decrease in BCL2 protein after overexpression of IFIT1, IFIT2 and IFIT3. The data represented as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are a representation of at least two independent studies

Fig. 3

HSP90 inhibition potentiates responses to immune checkpoint blockade in vivo. a Treatment schedule: Treatment with ganetespib and anti-CTLA begun at the same time. Ganetespib was administered at 100 mg/kg per mouse once a week and anti-CTLA4 at 100 µg/mouse every 3 days. n = 8 mice. b Tumor volumes across treatment groups over time. c Survival of animals depicted by Kaplan–Meier curves. n = 8 mice. Vehicle = Solvent+Isotype antibody control. Mice were sacrificed when moribund or when tumor volume reached 1000 mm³ or tumors developed ulceration >3mm in diameter. The data represented as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by two way ANOVA. Data are a representation of at least two independent studies

Fig. 4

Combining HSP90 inhibition and anti-CTLA4 enhances CD8 T-cell function. a Treatment schedule: ganetespib was administered at 100 mg/kg per mouse once a week and anti-CTLA4 at 100 μg per mouse every 3 days. Two doses of ganetespib and 4 doses of anti-CTLA4 were administered. At day 18, mice were sacrificed, spleens harvested and processed, tumors were harvested and processed for RNA and immune cell infiltration profiled via flow cytometry. b Absolute number of CD8 T cells infiltrating the tumor normalized to tumor weight. c Absolute number of T regulatory cells infiltrating tumor normalized to tumor weight. d Ratio of CD8 T cells to Tregs infiltrating the tumor. e Absolute number of granzyme A positive CD8 T cells normalized to tumor weight. f Absolute number of granzyme B positive CD8 T cells normalized to tumor weight. n = 9 mice. g ELISPOTS showing gp100 and P15E-specific cells from splenocytes of treated mice. The data represented as mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one way ANOVA for B-F or paired two tailed Student’s t-test for G. Data are a representation of at least two independent studies