Effective combinatorial immunotherapy for castration-resistant prostate cancer

  • A Corrigendum to this article was published on 04 May 2017

Abstract

A significant fraction of patients with advanced prostate cancer treated with androgen deprivation therapy experience relapse with relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC)1. Immune checkpoint blockade using antibodies against cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1/programmed cell death 1 ligand 1 (PD1/PD-L1) generates durable therapeutic responses in a significant subset of patients across a variety of cancer types2. However, mCRPC showed overwhelming de novo resistance to immune checkpoint blockade3,4,5, motivating a search for targeted therapies that overcome this resistance. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumour immune evasion6. The abundance of circulating MDSCs correlates with prostate-specific antigen levels and metastasis in patients with prostate cancer7,8,9. Mouse models of prostate cancer show that MDSCs (CD11b+Gr1+) promote tumour initiation10 and progression11. These observations prompted us to hypothesize that robust immunotherapy responses in mCRPC may be elicited by the combined actions of immune checkpoint blockade agents together with targeted agents that neutralize MDSCs yet preserve T-cell function. Here we develop a novel chimaeric mouse model of mCRPC to efficiently test combination therapies in an autochthonous setting. Combination of anti-CTLA4 and anti-PD1 engendered only modest efficacy. Targeted therapy against mCRPC-infiltrating MDSCs, using multikinase inhibitors such as cabozantinib and BEZ235, also showed minimal anti-tumour activities. Strikingly, primary and metastatic CRPC showed robust synergistic responses when immune checkpoint blockade was combined with MDSC-targeted therapy. Mechanistically, combination therapy efficacy stemmed from the upregulation of interleukin-1 receptor antagonist and suppression of MDSC-promoting cytokines secreted by prostate cancer cells. These observations illuminate a clinical path hypothesis for combining immune checkpoint blockade with MDSC-targeted therapies in the treatment of mCRPC.

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Figure 1: Strong combination synergy by ICB with cabozantinib or BEZ235 in mCRPC.
Figure 2: Cabozantinib and BEZ235 attenuate MDSC frequency and immunosuppressive activity in the tumour microenvironment.
Figure 3: Cabozantinib and BEZ235 inhibit PI3K signalling and modulate MDSC-regulating cytokines.
Figure 4: Gr-MDSCs inversely correlate with CD8+ infiltrating T cells in human PCa.

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Acknowledgements

We thank N. Feng for advice on preclinical drug dosing; X. Xu and K. O’Connor for technical support on mES cells and chimaera generation; P. Jones and C. Carroll for providing 19F NMR spectroscopy data; K. Zhao, Z. Xu, and Z. Fang for animal husbandry; F. Giancotti for suggestions on manuscript writing; A. Varma and J. W. Han for technical assistance; G. Wang, E.-J. Jin, P. Dey, and all members of the DePinho laboratory for a variety of technical support and suggestions. The project was supported by U01CA141508 (R.A.D.), P01CA117969 (R.A.D.), Department of Defense Prostate Cancer Research Program (PCRP) Idea Development Award–New Investigator Option W81XWH-14-1-0576 (X.L.), National Institutes of Health grant R44HL072614 (D.Y.M.), and the Clayton & Modesta Williams Cancer Research Fund (R.A.D.). Facility-based experimental support was provided by the Small Animal Imaging Facility (C. Kingsley, V. Tran, K. Maldonado, C. Kaffes), Flow Cytometry and Cellular Imaging Core Facility (J. Burks, D. Mak, and K. Dwyer), and Laboratory Animal Genetic Services (F. Benavides) at The University of Texas MD Anderson Cancer Center (Cancer Center Support Grant P30CA016672).

Author information

X.L., R.A.D., Y.A.W., and J.W.H. conceived the project and discussed experiments; J.W.H. designed the methodology for, and oversaw, the chimaeric modelling; X.L., E.P., and X.S. acquired and analysed the data; P.D. performed mouse genotyping; X.S., P.D., and Q.C. performed histology staining; P.T. collaborated on providing fresh human samples; S.J. oversaw and performed animal colony management; D.Y.M. provided study drug SX-682 and technical assistance for its use; J.A.Z. suggested MDSC experiments with SX-682 in the model; P.S. provided key suggestions on experiments; Y.A.W. and R.A.D. supervised the research; X.L., R.A.D., Y.A.W., and D.J.S. wrote the manuscript.

Correspondence to Y. Alan Wang or Ronald A. DePinho.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks D. Gabrilovich, M. Galsky, R. Madan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Chimaeric modelling as an efficient approach to generating spontaneous metastatic PCa.

a, Comparison of probability of obtaining PCa-bearing males with CPPSML genotype in a litter, through breeding or chimaeric modelling. In chimaeric modelling, >75% coat colour contributed by injected mES cells (mESC in the figure) is defined as positivity for chimaera. b, c, Predicted and experimental results for PCR–RFLP genotyping of the H2 locus from several mouse strains and two mES cell lines (JM8 is a standard mES cell line derived from C57BL/6 strain as control; Ep61 is also known as JH61). Red asterisk in b and yellow highlight wells in c indicate that the H2 haplotyte for JH61 is H2b, the same as the C57BL/6 strain. d, SSLP marker analysis of the region on chromosome 17 flanking the H2 complex locus (34–46 Mb), showing that JH61 has 100% C57BL/6 background in the H2 locus, identical to the standard C57BL/6 mES cell line JM8. e, Experimental steps for generating the CPPSML chimaeras. f, Fluorescence images of prostate, draining lymph node (LN), and lung from a representative chimaera at 3 months old. GFP+ signals indicate the presence of metastasis to lymph nodes and disseminated tumour cells and micrometastasis in lung. Scale bars: prostate, 5 mm; lymph node and lung, 1 mm. g, Fluorescence microscopy and H&E image of snap-frozen prostate tumour from chimaera showing that the GFP+ area corresponds to adenocarcinoma and the GFP area corresponds to normal host cells. Scale bar, 500 μm. h, Immunohistochemical staining showing the expansion of both CK8+ luminal lineage and CD5+ basal lineage in the prostate tumour formed in CPPSML chimaera. Scale bar, 50 μm. Source data

Extended Data Figure 2 Experimental design for preclinical therapy of mCRPC in CPPSML chimaeras.

a, Significant yet transient survival benefit by castration followed by diet admixed with enzalutamide (50 mg per kg diet) in PB-Cre+ PtenL/L p53L/L Smad4L/L mice (n = 40 and 18, respectively). ****P < 0.0001, log-rank test. b, Record of assignment for drug trials showing the time range of prostate tumour formation in the CPPSML chimaera. c, Representative CPPSML chimaera with primary CRPC, lymph node metastasis, and micrometastasis in lung. Scale bars: prostate, 5 mm; lymph node and lung, 1 mm. AP, DLP, and VP denote anterior, dorsolateral, and ventral prostate lobes, respectively. d, Experimental flow for creating mCRPC cohorts and preclinical testing of monotherapy and combination therapy, followed by tumour characterization. e, Representative images of prostate tumours with H&E staining, and GFP+ tumour cells in the lymph node and lung. Scale bars: prostate, 5 mm; lymph node and lung, 1 mm; H&E, 200 μm. Source data

Extended Data Figure 3 Significant combination efficacy by cabozantinib and ICB observed in chimaeras generated from JH58.

a, Experimental design for JH58 chimaeras, similar to the JH61 chimaera experiments. b, Longitudinal MRI images from representative chimaeras in control or combination cohorts. Red contour denotes area of prostate tumour. c, Strong anti-tumour effect by combination therapy in JH58 chimaeras shown by prostate tumour mass, lymph node metastasis scores and lung micrometastasis number (n = 3, biological replicates). **P < 0.01, Student’s t-test. d, e, Quantification of tumour cell proliferation by Ki67 immunohistochemistry (n = 4, biological replicates) with representative images. Anterior prostate (AP) and dorsolateral prostate (DLP) were quantified separately. f, g, Quantification of tumour cell apoptosis by cleaved caspase 3 immunohistochemistry (n = 5, biological replicates) with representative images. Scale bar, 100 μm. In c, d, and f, data represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control using Student’s t-test. Source data

Extended Data Figure 4 Combination efficacy by Gr1 neutralizing antibody with ICB.

a, Dasatinib, but not cabozantinib or BEZ235, significantly reduced the frequency of infiltrating T cells in CRPC of CPPSML mice (n = 4, biological replicates). Data represent mean ± s.e.m. *P < 0.05, Mann–Whitney U-test. b, Frequency of Gr-MDSCs and Mo-MDSCs in CPPSML prostate tumours (n = 13, biological replicates). c, Mass and representative whole-organ and H&E images of prostate tumours from CPPSML chimaeras induced to develop CRPC and treated with 1 month of control IgG, ICB (anti-CTLA4 plus anti-PD1 antibodies), anti-Gr1 neutralizing antibody, or combination of ICB and anti-Gr1 (n = 4, biological replicates). Scale bars: 3 mm for organ images, 200 μm for H&E images. In b and c, data represent mean ± s.d. *P < 0.05, **P < 0.01, ****P < 0.0001, #P > 0.05, Mann–Whitney U-test. Source data

Extended Data Figure 5 Characterization of the effect of drugs on MDSCs.

a, Comparison of in vitro sensitivity to Dasa by MDSCs, CD8+ T cells, and GFP+ cancer cells isolated from CRPC of CPPSML mice. Cell viability was measured 24 h after the start of drug treatment using the WST-1 assay. IC50 values are indicated. b, Comparison of in vitro sensitivity to BEZ, Cabo, and Dasa by MDSCs isolated from CRPC of CPPSML mice. The assay was performed in RPMI1640 supplemented with 10% FBS and 10 ng ml−1 GM-CSF (n = 2, biological replicates). c, Comparison of in vitro sensitivity to BEZ, Cabo, and Dasa by MDSCs isolated from CRPC of CPPSML mice. The assay was performed in RPMI1640 supplemented with 10% FBS, 10 ng ml−1 GM-CSF, and pre-conditioned for 12 h by PCa cell lines established from the CPPSML model (n = 2, biological replicates). d, Representative CFSE flow cytometry histograms showing the effect on in vitro T-cell proliferation by MDSCs isolated from CRPC of CPPSML mice treated with the indicated drugs. Position of CFSE peaks can be used to denote the T-cell division times. e, Representative CFSE flow cytometry histograms showing the effect of Cabo, BEZ, and Dasa on in vitro T-cell proliferation. Source data

Extended Data Figure 6 Cabozantinib and BEZ235 inhibit PI3K signalling in prostate tumour and intratumoural MDSCs.

a, Mouse phospho-RTK array measuring phospho-RTK activity in prostate tumours with indicated treatments. Numerals 1–5 represent pEGFR, pErbB2, pErbB3, pAxl, and pPDGFRα, respectively (n = 2, biological replicates). b, c, Reduced pS6 signal in intratumoural MDSCs by Cabo and BEZ treatment, revealed by immunofluorescent co-staining of pS6 and Gr-1 (n = 3, biological replicates). Scale bar, 100 μm. d, WST-1 assay showing that co-transfection of active ERK2 and p70S6K proteins mediated the resistance of MDSCs isolated from CPPSML tumours to the cytotoxicity by Cabo (1.5 μM) or BEZ (0.15 μM). The assay was performed in RPMI1640 supplemented with 10% FBS and 10 ng ml−1 GM-CSF (n = 3, biological replicates). e, WST-1 assay similar to d, but performed in RPMI1640 supplemented with 10% FBS, 10 ng ml−1 GM-CSF, and pre-conditioned for 12 h by PCa cell lines established from the CPPSML model (n = 3, biological replicates). In ce, data represent mean ± s.d. ***P < 0.001, Student’s t-test. Source data

Extended Data Figure 7 Cabozantinib or BEZ235 suppress secretion by PCa cells of several cytokines that promote MDSC activity.

a, Quantification of intratumoural cytokine levels in CRPC chimaera tumours with indicated treatment using cytokine array (n = 2, biological replicates). Numerals 1–9 represent CCL5, CCL12, CCL21, CD40, CD142, HGF, IGFBP-6, IL-1ra, and VEGF, respectively. b, Quantification of intratumoural cytokine levels in Dasa + ICB combination-treated CPPSML chimaera CRPC with mouse cytokine assay, with image and relative intensity of the numbered cytokines shown (n = 2, biological replicates). c, Experimental design for MDSC culture in the presence of PCa conditioned medium. d, Cytokine array results for conditioned medium from CPPSML PCa cell lines treated with vehicle, Cabo (1 μM), or BEZ (1 μM) for 12 h (n = 2, biological replicates). Boxed cytokine is CCL5. e, Effect of supplementation of individual cytokines to the conditioned medium from PCa cell lines treated with Cabo (1 μM) or BEZ (1 μM) on Arg1, Cybb, Ncf1, and Ncf4 from cultured MDSCs (n = 3, biological replicates). f, g, Chemical structure and synthesis of allosteric CXCR1/2 antagonist SX-682 (Syntrix Biosystems). For details, please refer to the corresponding section in Methods. In b, e, and f, data represent mean ± s.d. Source data

Extended Data Figure 8 Detailed cell population annotation in SPADE tree.

a, SPADE tree coloured by the median intensity of individual markers (indicated above colour bar) to facilitate the assignment of tree branches to individual cell populations (shown on the top of each plot) (n = 12, biological replicates). b, Surface markers of different immune subpopulations representing small branches of the SPADE tree.

Extended Data Figure 9 Model depicting the combination therapy strategy in treating mCRPC.

As demonstrated in the CPPSML chimaera model, targeted therapy with agents that inhibit MDSC infiltration frequency and immunosuppressive activity can synergize with ICB to invigorate T-cell immunity in the prostate tumour microenvironment and thus impair CRPC progression.

Extended Data Table 1 Additional information on mouse model, clinical samples, and methodology

Supplementary information

Supplementary Figure 1

This file contains the Source data for western blot as follows: (a) Source gel images for Figure 3b and (b) Source gel images for Figure 3c. (PDF 750 kb)

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Lu, X., Horner, J., Paul, E. et al. Effective combinatorial immunotherapy for castration-resistant prostate cancer. Nature 543, 728–732 (2017). https://doi.org/10.1038/nature21676

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