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. 2020 Sep;10(9):1374-1387.
doi: 10.1158/2159-8290.CD-19-1352. Epub 2020 May 8.

Chromatin Regulator CHD1 Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer

Di Zhao #  1   2 , Li Cai #  1 , Xin Lu #  1   3 , Xin Liang  1   4 , Jiexi Li  1 , Peiwen Chen  1 , Michael Ittmann  5 , Xiaoying Shang  1 , Shan Jiang  6 , Haoyan Li  2 , Chenling Meng  2 , Ivonne Flores  6 , Jian H Song  4 , James W Horner  6 , Zhengdao Lan  1 , Chang-Jiun Wu  6 , Jun Li  6 , Qing Chang  7 , Ko-Chien Chen  1 , Guocan Wang  1   4 , Pingna Deng  1 , Denise J Spring  1 , Y Alan Wang  8 , Ronald A DePinho  8
Affiliations

Chromatin Regulator CHD1 Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer

Di Zhao et al. Cancer Discov. 2020 Sep.

Abstract

Genetic inactivation of PTEN is common in prostate cancer and correlates with poorer prognosis. We previously identified CHD1 as an essential gene in PTEN-deficient cancer cells. Here, we sought definitive in vivo genetic evidence for, and mechanistic understanding of, the essential role of CHD1 in PTEN-deficient prostate cancer. In Pten and Pten/Smad4 genetically engineered mouse models, prostate-specific deletion of Chd1 resulted in markedly delayed tumor progression and prolonged survival. Chd1 deletion was associated with profound tumor microenvironment (TME) remodeling characterized by reduced myeloid-derived suppressor cells (MDSC) and increased CD8+ T cells. Further analysis identified IL6 as a key transcriptional target of CHD1, which plays a major role in recruitment of immunosuppressive MDSCs. Given the prominent role of MDSCs in suppressing responsiveness to immune checkpoint inhibitors (ICI), our genetic and tumor biological findings support combined testing of anti-IL6 and ICI therapies, specifically in PTEN-deficient prostate cancer. SIGNIFICANCE: We demonstrate a critical role of CHD1 in MDSC recruitment and discover CHD1/IL6 as a major regulator of the immunosuppressive TME of PTEN-deficient prostate cancer. Pharmacologic inhibition of IL6 in combination with immune checkpoint blockade elicits robust antitumor responses in prostate cancer.This article is highlighted in the In This Issue feature, p. 1241.

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Conflict of interest statement

Conflict of interest: R.A.D. is a founder, advisor and/or director of Tvardi Therapeutics, Inc., Nirogy Therapeutics, Inc., and Asylia Therapeutics, Inc. which are focused on cancer, fibrosis and/or inflammation. The work of these entities is not related to the science of this manuscript. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1.
Figure 1.. Genetic deletion of Chd1 inhibits development of PTEN-null prostate cancer
(A) GEM model design: Conditional knockout alleles of Chd1Loxp, PtenLoxp, and Smad4Loxp were crossed with prostate-specific PB-Cre and Rosa-mTmG to establish a prostate-specific PtenChd1 or PtenSmad4Chd1 knockout prostate cancer mouse model. Ptenpc−/−, PB-Cre PtenL/L; Chd1pc−/−, PB-Cre Chd1L/L; PtenChd1pc−/−, PB-Cre PtenL/L Chd1L/L; PtenSmad4pc−/−, PB-Cre PtenL/L Smad4L/L; PtenSmad4pc−/−, PB-Cre PtenL/L Smad4L/L; PtenSmad4Chd1pc−/−, PB-Cre PtenL/L Smad4L/L Chd1L/L. (B-C) Prostate tumor MRI and tumor volume of Ptenpc−/− and PtenChd1pc−/− mice at 12 months of age. (D) H&E staining of prostate tumors from 7-month old Ptenpc−/− and PtenChd1pc−/− mice. AP: anterior prostate; DLP: dorsal-lateral prostate. Scale bar: 200 μm. (E) IHC staining of CHD1 and phospho-AKT (Ser473) markers and (F) quantification of Ki67+ cells of Ptenpc−/− vs. PtenChd1pc−/− prostate tumors. Scale bar: 100 μm. (G) Kaplan-Meier survival curve of wild type, Chd1pc−/−, Ptenpc−/− and PtenChd1pc−/− mice. (H-I) MRI and tumor volume of prostate tumors from PtenSmad4pc−/− mice with or without Chd1 deletion at 4 months of age. (J) Lymph node metastasis rate and (K) Kaplan-Meier survival curve of PtenSmad4pc−/− and PtenSmad4Chd1pc−/− mice.
Figure 2.
Figure 2.. CHD1 promotes an immunosuppressive TME in prostate cancer
(A) Top 15 down-regulated pathways in PtenChd1pc−/− prostate tumors. Pathways highlighted in red are immune response related pathways. NES: normalized enrichment score. (B-D) Immunoprofiling of Ptenpc−/− vs. PtenChd1pc−/− prostate tumors using CyTOF. (B,C) viSNE plots colored by relative expression of CyTOF markers, with populations indicated, and (D) quantification of each tumor infiltrating immune cell population. (E,F) Immunoprofiling of PtenSmad4pc−/− vs. PtenSmad4Chd1pc−/− prostate tumors using CyTOF. (E) viSNE plots and (F) quantification of tumor infiltrating immune cell populations. PS, PtenSmad4pc−/−; PSC, PtenSmad4Chd1pc−/−
Figure 3.
Figure 3.. CHD1 controls MDSC recruitment
(A,B) Immunofluorescence staining and quantification of MDSC marker (Ly6G) and CD8+ T cell marker (CD8a) in Ptenpc−/− vs. PtenChd1pc−/− prostate tumors. (C) CHD1 expression correlates with MDSC enrichment in human prostate tumors. (D) Correlation analysis of CHD1 and MDSC marker CD15 expression in TCGA dataset. (E,F) Correlation analysis of CD8+ T cells infiltration and CHD1 expression in human prostate cancer samples (n = 72; r = −0.273; p = 0.02). CHD1 expression: Low or High. CD8 score: 0-2. Scale bar: 100 μm. (G) Western blot of CHD1-depleted or control murine prostate cancer cells. (H,I) In vitro migration assay of MDSCs and T cells in the conditioned medium collected from CHD1-depleted or control murine prostate cancer cells.
Figure 4.
Figure 4.. IL-6 is a direct target gene of CHD1
(A) Venn diagram of CHD1 directly regulated genes identified by ChIP-seq and differential expression genes in CHD1 depletion PC-3 or murine prostate tumors. The overlapping 11 genes are considered direct target genes of CHD1. (B) GSEA analysis of wild type, Ptenpc−/− vs. PtenChd1pc−/− prostate samples indicates the IL-6-Stat3 pathway regulated by PTEN-CHD1 axis. (C) ChIP-seq in Pten deletion prostate cancer cells revealed binding peaks of CHD1 on IL-6 gene promoter region. CHD1-ChIP-1: CHD1 antibody from CST; CHD1-ChIP-2: CHD1 antibody from Bethyl. (D,E) ChIP-seq analysis of top CHD1 binding motif (TGAG/CTCA), which is conserved in human and murine IL-6 promoter. (F,G) Correlation analysis of CHD1 expression and IL-6 or phospho-Stat3 level in human prostate tumors (TCGA data)
Figure 5.
Figure 5.. IL-6 serves as a key mediator for MDSC recruitment induced by CHD1
(A) IL-6 gene expression of Ptenpc−/− vs. PtenChd1pc−/− prostate tumors determined using qPCR. (B) ELISA assay of IL-6 in Ptenpc−/− vs. PtenChd1pc−/− prostate tumors. (C) Luciferase assay reveals CHD1 directly regulates the activation of IL6 promoter. (D) Luciferase assay with wild type or depleted CHD1 binding motif in the IL6 promoter region. (E,F) In vitro MDSC migration assay in the presence of IL6 recombinant proteins in CHD1 depletion conditioned medium (E) or IL-6 or IL-6R inhibitors in wild type conditioned medium (F).
Figure 6.
Figure 6.. Synergistic anti-tumor effect of CHD1/IL-6 inhibition in combination with ICI in prostate cancer
(A) Schematic illustrating the generation of inducible-shCHD1 DX1 cells and orthotopic prostate cancer mouse model, followed by the treatment with Doxycycline and anti-PD1. (B) Tumor volume was measured by MRI after 2 weeks of treatment. (C-D) The anti-tumor effects of combinatorial IL-6 inhibition and anti-PD1/CTLA4 dual blockade were evaluated in the “CPPSML” chimeric prostate cancer model. Tumor growth was monitored by MRI bi-weekly; MRI images (C) and volumes (D) after 6 treatments are shown here. (E-F) MRI imaging and tumor volume after single, dual or triple blockades of IL-6/PD1/CTLA-4 in the PtenSmad4pc−/− prostate cancer GEM model. (G) viSNE plots of tumor infiltrating immune cells in PtenSmad4pc−/− prostate tumors treated with IgG or triple blockades of IL-6/PD1/CTLA-4.
Figure 7.
Figure 7.. CHD1 contributes to the remodeling of the TME and resistance to ICI
In PTEN-loss prostate cancer cells, CHD1 is stabilized and interacts with active epigenetic marker trimethylation of histone H3 at lysine 4 (H3K4me3) (17). IL-6 serves as a direct target gene of CHD1 and contributes to recruit immunosuppressive MDSCs, resulting in the inhibition of CD8+ T cells in prostate tumor. Disruption of the CHD1-IL-6 axis suppresses MDSC infiltration and boosts intratumor CD8+ T cells. In combination with immune checkpoint inhibition, IL-6 inhibition shows durable therapeutic effects on PTEN-deficient prostate cancer.
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