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Autophagy inhibition by targeting PIKfyve potentiates response to immune checkpoint blockade in prostate cancer

Nature Cancer volume 2, pages 978–993 (2021)Cite this article

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Abstract

Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I–cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The antitumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon-γ pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve knockdown recapitulated ESK981’s antitumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in patients with advanced prostate cancer and be an effective treatment strategy alone or in combination with immunotherapies.

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Fig. 1: ESK981 inhibits the growth of prostate cancer cells in vitro and is associated with a vacuolization morphology.
Fig. 2: ESK981 inhibits the growth of diverse preclinical models of prostate cancer in vivo.
Fig. 3: ESK981 induces the accumulation of autophagosomes in prostate cancer cells.
Fig. 4: ESK981 induces the accumulation of lysosomes through inhibition of autophagic flux in prostate cancer cells.
Fig. 5: ESK981 activates an antitumor immune response in immune-competent murine prostate cancer models.
Fig. 6: ESK981 potentiates the effect of anti-PD-1 immunotherapy in immune-competent murine prostate cancer models.
Fig. 7: Identification of lipid kinase PIKfyve as the target of ESK981-induced effects on autophagy and CXCL10 levels.
Fig. 8: Genetic inhibition of Pikfyve potentiates the therapeutic benefit of anti-PD-1 immunotherapy in immune-competent murine models.

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Data availability

Raw RNA-seq data have been deposited at the NCBI Gene Expression Omnibus (GSE174644). Further information and requests for resources and reagents should be directed to the corresponding author. All requests for raw and analyzed data and materials will be reviewed promptly by the corresponding author to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a material transfer agreement. Source data are provided with this paper.

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Acknowledgements

We thank M. Trierweiler and S. Zelenka-Wang for histological sample processing and IHC, as well as undergraduate student K. Johnson for technical assistance. This work was supported by a Prostate Cancer Foundation Challenge Award, NCI Prostate SPORE Grant P50CA186786, Department of Defense PC130151P1 (to N.M.N. and A.M.C.) and NIH grant GM131919 (to D.J.K.). A.M.C. is an NCI Outstanding Investigator (R35CA231996), Howard Hughes Medical Institute Investigator, A. Alfred Taubman Scholar and American Cancer Society Professor. Y.Q. and J.C.T. are supported by Prostate Cancer Foundation Young Investigator awards. L.X. is supported by a Department of Defense Postdoctoral Award (W81XWH-16-1-0195). E.-L.E. was supported by the Academy of Finland.

Author information

Authors and Affiliations

  1. Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, USA

    Yuanyuan Qiao, Jae Eun Choi, Jean C. Tien, Stephanie A. Simko, Josh N. Vo, Andrew D. Delekta, Lisha Wang, Lanbo Xiao, Nathan B. Hodge, Parth Desai, Sergio Mendoza, Kristin Juckette, Alice Xu, Fengyun Su, Rui Wang, Xuhong Cao, Xiao-Ming Wang, Xiaoju Wang, Javed Siddiqui, Ester Fernandez-Salas, Stephanie J. Ellison & Arul M. Chinnaiyan

  2. Department of Pathology, University of Michigan, Ann Arbor, MI, USA

    Yuanyuan Qiao, Jean C. Tien, Thekkelnaycke Rajendiran, Xiao-Ming Wang, Weiping Zou & Arul M. Chinnaiyan

  3. Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA

    Yuanyuan Qiao, Weiping Zou & Arul M. Chinnaiyan

  4. School of Medicine, University of California, San Diego, La Jolla, CA, USA

    Jae Eun Choi

  5. Michigan Regional Comprehensive Metabolomics Resource Core, Ann Arbor, MI, USA

    Thekkelnaycke Rajendiran & Tanu Soni

  6. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Josh N. Vo

  7. Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI, USA

    Xuhong Cao & Arul M. Chinnaiyan

  8. Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, USA

    Jiali Yu, Ilona Kryczek & Weiping Zou

  9. School of Pharmacy, Jinan University, Guangzhou, China

    Zhen Wang & Ke Ding

  10. CNRS, Laboratoire de Biogenèse Membranaire, Université de Bordeaux, Bordeaux, France

    Amélie Bernard

  11. Life Sciences Institute, Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Amélie Bernard & Daniel J. Klionsky

  12. Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Nora M. Navone

  13. Institute of Biomedicine, University of Turku, Turku, Finland

    Eeva-Liisa Eskelinen

  14. Karmanos Cancer Institute, Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA

    Elisabeth I. Heath

  15. Department of Urology, University of Michigan, Ann Arbor, MI, USA

    Arul M. Chinnaiyan

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  1. Yuanyuan Qiao
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  34. Arul M. Chinnaiyan
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Contributions

Y.Q. and A.M.C. participated in the planning, initiation and overall analysis of data, as well as writing, reviewing and editing of the manuscript. Y.Q., S.A.S., A.D.D., N.B.H., P.D. and S.M. performed the in vitro and in vivo experiments. J.C.T., J.E.C., K.J. and A.X. participated in the in vivo experiments. Y.Q., T.R. and T.S. participated in the lipidomics experimental design and data analysis. Z.W. and K.D. participated in execution of the chemical synthesis of ESK981. L.W., X.-M.W. and J.S. performed the histological sample preparation, staining and interpretation of RNA ISH results. L.X. helped with the CRISPR Atg5 knockout design. X.W. assisted with the ELISA experiments. X.C., F.S., R.W. and J.N.V. performed the RNA-seq library preparation, sequencing and data analysis. J.Y., I.K. and J.E.C. participated in the flow cytometry analysis. A.B. and D.J.K. participated in the yeast experiments and data interpretation. E.-L.E. performed the electron microscopy analysis. E.-L.E. and D.J.K. participated in the autophagy data interpretation. N.M.N. provided the PDX models. S.J.E. participated in writing and preparation of the manuscript. W.Z. participated in the immune checkpoint blockade experimental design and data interpretation. E.F.-S., E.I.H. and A.M.C. provided project oversight for clinical trial design and review based on the interpretation of the preclinical data.

Corresponding author

Correspondence to Arul M. Chinnaiyan.

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Competing interests

The University of Michigan has filed a disclosure on the findings based on this study. A.M.C. and Y.Q. are named as co-inventors on the disclosure. Esanik Therapeutics licensed ESK981 from Teva Pharmaceuticals. A.M.C. is a co-founder of Esanik Therapeutics and serves on its scientific advisory board. Neither Esanik Therapeutics nor Teva Pharmaceuticals was involved in the design or approval of this study, nor was this study funded by them. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Cory Abate-Shen, Thorbald van Hall and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 ESK981 blocks cell growth, induces cell cycle arrest, and decreases cellular invasion.

a-b, Representative crystal violet staining for a long-term survival assay of a panel of prostate cell lines at various concentrations of ESK981, crizotinib, or cabozantinib. c, Cell cycle analysis was measured after 72 hours of increasing concentrations of ESK981 treatment in indicated prostate cancer cell lines. Ctrl, control. d, Cell cycle analysis of VCaP cells that were treated with the indicated compounds for 72 hours. Cabo, cabozantinib; Crizo, crizotinib; Enza, enzalutamide; ESK, ESK981. e, Matrigel invasion assay of various prostate cancer cell lines that were treated with the indicated concentrations of ESK981. The percentage invasion was quantified with a fluorescent plate reader. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

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Extended Data Fig. 2 ESK981 inhibits the growth of diverse preclinical models of prostate cancer in vivo.

a, Schematic illustration of the VCaP CRPC mouse xenograft experimental design. To generate castration-resistant VCaP, parental VCaP cells were injected subcutaneously into both flanks of intact male mice. When average VCaP tumors reached 200 mm3, mice were surgically castrated and VCaP tumors regressed due to loss of androgen. Castration-resistant VCaP tumors developed as VCaP tumors grew back to the size of pre-castration. Castration-resistant VCaP tumors were then randomized into three groups and treated with vehicle, 30 mg/kg, or 60 mg/kg ESK981 p.o., oral gavage. b, Representative IHC images for proliferation marker Ki67 are shown after treatment with the indicated drugs for five days in VCaP tumors (left). Quantification of positive Ki67 percentage is shown on the right (right). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. N = 4 tumors per group. P-value indicated. c, Representative individual tumors from vehicle and ESK981 groups in AR+ and ERG+ prostate PDX MDA-PCa-146-12 (left). Representative IHC showing Ki67 staining for vehicle and 30 mg/kg ESK981 groups of MDA-PCa-146-12 tumors (right) from three independent experiments. d, Representative individual tumors from vehicle and ESK981 groups of DU145 tumors (left). Representative IHC showing Ki67 staining for the vehicle and 30 mg/kg ESK981 groups of DU145 tumors (right) from three independent experiments.

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Extended Data Fig. 3 Renal function, liver function, and histopathological evaluation of ESK981-treated xenografts.

a, Castration-resistant VCaP tumors were established according to Extended Data Fig. 2a. Tumor-bearing mice were divided into vehicle and ESK981 50 mg/kg groups, and tumor volumes were monitored twice per week for six weeks. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM at day 25. N = number of tumors and P-value indicated. b, The percent body weights of VCaP tumor-bearing mice were monitored daily throughout this study. Data were presented as mean ± SEM. N = number of mice. c, The weight of VCaP tumors from vehicle (n = 18 tumors) and ESK981 50 mg/kg (n = 10 tumors) were measured at the end of this study. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. P-value indicated. d, Blood chemistry was evaluated for renal and liver functions in non-tumor-bearing and VCaP tumor-bearing mice in vehicle and 50 mg/kg ESK981 treatment groups. e, Representative histological sections showing H&E staining for various organs taken from vehicle- or ESK981-treated mice from three independent experiments. f, Representative histological sections showing H&E staining for tumors taken from vehicle- or ESK981-treated mice from three independent experiments.

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Extended Data Fig. 4 ESK981 robustly induces autophagosome levels and is dependent on ATG5 for its effects.

a, DU145 cells with the indicated drug treatment for 24 hours. Autophagosome induction activity was visualized by CYTO-ID® assay from three independent experiments. Rapa, rapamycin. b, VCaP cells were treated with 300 nM ESK981 for the indicated time points, and LC3 protein levels were assessed by western blot from three independent experiments. c, VCaP cells were treated with ESK981 (ESK), crizotinib (Crizo), and cabozantinib (Cabo) at the indicated concentrations. Protein levels of LC3 were examined after 24 hours of treatment from three independent experiments. d, Protein levels of Atg8 in yeast prd5Δ cells after ESK981 (ESK) or cabozantinib (Cabo) treatment under nitrogen deprivation conditions. NT, no treatment. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. P value indicated. e, Protein levels of indicated protein post various siRNA knockdown in VCaP and LNCaP cells with or without 300 nM ESK981 or 1 µM sunitinib treatment for 24 hours from three independent experiments.

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Extended Data Fig. 5 ESK981 upregulates CXCL10 expression in human prostate cancer cells and inhibits autophagy in murine Myc-CaP prostate cancer cells.

(a) CXCL10 protein levels measured by ELISA in conditioned media from VCaP cells treated with ESK981 or various autophagy inducers for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (b) CXCL10 mRNA levels measured by quantitative PCR (qPCR) in VCaP, PC3, and DU145 cells with the indicated treatment for 24 hours. IFNγ, interferon gamma. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (c) IC50 of ESK981, crizotinib, and cabozantinib determined in Myc-CaP cells. (d) Protein levels of LC3 after 50 nM, 100 nM, and 300 nM ESK981 treatment for 24 hours in Myc-CaP cells from three independent experiments. (e) Ratio of GFP/RFP signal in Myc-CaP GFP-LC3-RFP-LC3∆G stable expressing cells with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. P-value indicated.

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Extended Data Fig. 6 Atg5 deletion blocks ESK981-induced vacuolization and CXCL10-mediated immune response.

(a) Myc-CaP wild-type (WT) and Atg5 knockout (Atg5 KO) cells were treated with increasing concentrations of ESK981 for 24 hours. Atg5 and LC3 levels were assessed by western blot from three independent experiments. GAPDH served as a loading control. (b) Representative morphology of vacuolization in Myc-CaP wild-type (WT) and Atg5 knockout (Atg5 KO) cells after treatment with control or 100 nM ESK981 for 24 hours from three independent experiments. (c) Autophagosome content of Myc-CaP WT and Atg5 KO cells were measured by CYTO-ID® assay after being treated with increasing concentrations of ESK981 for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (d) Mouse cytokine array using Myc-CaP WT and Atg5 KO cell supernatant after treatment with 10 ng/ml mouse interferon gamma (mIFNγ) or mIFNγ + 100 nM ESK981 for 24 hours. Differential expression candidate dots are highlighted by boxes. (e) Mouse CXCL10 protein levels were measured by ELISA in Myc-CaP WT and Atg5 KO conditioned medium with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (f) mRNA levels of Cxcl10 and Cxcl9 were measured by qPCR in Myc-CaP WT and Atg5 KO cells with 50 nM or 100 nM ESK981 and 10 ng/ml mIFNγ treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

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Extended Data Fig. 7 Transcriptomic analysis of Myc-CaP tumors treated with ESK981 in combination with anti-PD-1 immunotherapy in FVB mice.

(a) Principal Component Analysis (PCA) of individual Myc-CaP tumors from indicated treatment groups based on variance-stabilizing transformation (vst) of read-count data. The vehicle and ESK981+anti-PD-1 combination groups form a relatively distinct cluster based on the first two principal components. (b) Volcano plot of differential gene expression analysis for groups treated with ESK981+anti-PD-1 versus vehicle. The horizontal dashed line corresponds to the FDR = 0.05. The vertical dashed lines correspond to log2FC >= 1 (up-regulation) or log2FC <= -1 (down-regulation). (c) Mouse Gene Set Enrichment Analysis (GSEA) with biological process gene ontology for groups treated with ESK981+anti-PD-1 versus vehicle. Top 10 gene sets are ordered by normalized enrichment score (NES). The top enriched categories are relevant to immune responses and inflammation. (d) Heatmap representation of top differentially expressed genes in groups treated with ESK981+anti-PD-1 versus vehicle (FDR <= 0.01, up or down-regulated by at least 2-fold). (e) Fragments per kilobase of exon model per million reads mapped (FPKM) of indicated targets from individual Myc-CaP tumors treated with vehicle (n = 10 tumors), ESK981 (n = 8 tumors), anti-PD-1 (n = 7 tumors), or ESK981+anti-PD-1 (n = 8 tumors). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. P-value indicated.

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Extended Data Fig. 8 ESK981 induces autophagosome formation and upregulates Cxcl10 expression in various murine cancer cell lines.

a, IC50 from cell viability assays of ESK981 in murine cancer cells of lung (Ae17, LLC), melanoma (B16F10), ovarian (ID8), pancreas (PAN02), renal (Renca), prostate (TRAMP-C2), and breast (4T1) lineages. Data were plotted as mean ± SEM from three independent experiments. b, Autophagosome content measured by CYTO-ID in indicated cell lines treated with control (Ctrl) or 300 nM ESK981 for 24 hours. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. P-value indicated. c, mRNA level of Cxcl10 in indicated cell lines treated with 10 ng/ml mIFNγ or mIFNγ plus 300 nM ESK981 for 24 hours. Data were analyzed by two-tailed unpaired t test from three (PAN02, Ae17) or four (ID8, B16F10, Renca, 4T1, TRAMP-C2, LLC) independent experiments and presented as mean ± SEM. P-value indicated.

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Extended Data Fig. 9 ESK981 sensitizes the murine breast cancer 4T1 model to anti-PD-1 immunotherapy.

a, Bioluminescent signaling images showing dorsal and ventral views of individual 4T1 tumor-bearing mice from indicated treatment groups. b, Bioluminescent quantification of total tumor burden from individual mice treated with vehicle (n = 5 mice), anti-PD-1 (n = 4 mice), ESK981 15 mg/kg (n = 5 mice), ESK981+anti-PD-1 (n = 5 mice). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. P-value indicated. c, Overall survival of 4T1-bearing mice treated with either anti-PD-1 (n = 15 mice) or ESK981 and anti-PD-1 combination (n = 15 mice).

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Extended Data Fig. 10 PIKfyve mediates a cellular vacuolization morphology in human prostate cancer cells and murine cancer cells, and Pikfyve loss induces accumulation of autophagosomes in various murine cancer cells.

a, Morphology of DU145 and PC3 cells after siNC, siPIKFYVE, siPIP5K1C, or siPIK3CA transfection from three independent experiments. b, mRNA levels of PIKFYVE, PIP5K1C, and PIK3CA were measured by qPCR after siRNA knockdown of indicated targets in DU145 and PC3 cells. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM from three independent experiments. P-value indicated. c, Morphological changes of TRAMP-C2, ID8, and Ae17 cells after siNC or siPikfyve transfection from three independent experiments. d, Autophagosome induction activity measured with CYTO-ID® assay in TRAMP-C2, ID8, and Ae17 cells after siRNA knockdown of Pikfyve. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM from four (TRAMP-C2 and ID8) and six (Ae17) independent experiments. P-value indicated.

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Qiao, Y., Choi, J.E., Tien, J.C. et al. Autophagy inhibition by targeting PIKfyve potentiates response to immune checkpoint blockade in prostate cancer. Nat Cancer 2, 978–993 (2021). https://doi.org/10.1038/s43018-021-00237-1

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