Immunotherapy is a treatment for many types of cancer, primarily due to deep and durable clinical responses mediated by immune checkpoint blockade (ICB) 1,2 . Prostate cancer is a notable exception in that it is generally unresponsive to ICB. The standard treatment for advanced prostate cancer is androgen-deprivation therapy (ADT), a form of castration (CTX). ADT is initially effective, but over time patients eventually develop castrationresistant prostate cancer (CRPC). Here, we focused on defining tumor-cell intrinsic factors that contribute to prostate cancer progression and resistance to immunotherapy.We analyzed cancer cells isolated from castration-sensitive and castration-resistant prostate tumors, and discovered that castration resulted in significant secretion of Interleukin-8 (IL-8) and it's likely murine homolog Cxcl15. These chemokines drove subsequent intra-tumoral infiltration with polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), promoting tumor progression.PMN-MDSC infiltration was abrogated when IL-8 was deleted from prostate cancer epithelial cells using CRISPR/Cas9, or when PMN-MDSC migration was blocked with antibodies against the IL-8 receptor CXCR2. Blocking PMN-MDSC infiltration in combination with anti-CTLA-4 delayed the onset of castration resistance and increased the density of polyfunctional CD8 T cells in tumors. Taken together, our findings establish castration-mediated IL-8 secretion and subsequent PMN-MDSC infiltration as a key suppressive mechanism in the
Immunotherapy with agents that block immune checkpoints is a mainstay of therapy for several common tumor types; so far, prostate cancer is not among those treated using this method. The observed lack of activity in prostate cancer is not due to a lack of testing; several agents have been evaluated both alone and in combination. Although several combination strategies show some promise, it appears likely that a greater understanding of the prostate cancer tumor microenvironment and baseline immune response will be required to optimize future treatment strategies.
23 24 post-castration. We used the MCRedAL prostate cancer cell line; an RFP expressing 66 version of the Myc-Cap cell line characterized by MYC overexpression 15 . Like human 67 prostate cancer, MCRedAL tumors are initially castration-sensitive (CS), but castration-68 resistance (CR) develops approximately 30 days after castration (Extended Data Fig. 1a). 69 Pre-and post-ADT tumor cells were sorted to > 96% purity (Extended Data Fig. 1b) and 70 analyzed ( Fig. 1a-b and Extended Data Fig. 1c). A number of cytokine and chemokine 71 transcripts were significantly up-regulated post-ADT ( Fig. 1b right), including Cxcl15, a 72 CXC chemokine with a conserved ELR motif (Extended Data Table 1), which is the likely 73 murine homolog of human IL-8 (CXCL8) [16][17][18][19] . qRT-PCR and ELISA assays confirmed the 74 upregulation of Cxcl15 post-ADT at the protein level (Extended Data Fig. 1d). In addition 75 to the chemokines above, GSEA revealed the upregulation of several pro-inflammatory 76 pathways post-ADT ( Fig. 1c). In vitro experiments using the human androgen-responsive 77 LNCaP cell line corroborated a role for these pro-inflammatory signals, showing that in 78 the absence of androgen, TNFα upregulated IL-8 expression in a dose-dependent 79 manner ( Fig. 1d left); while AR signaling in the absence of inflammation did not affect IL-80 8 expression ( Fig. 1d right). These data led to the hypothesis that AR signaling directly 81 suppresses IL-8 expression in prostate cancer cells. We performed in silico ChIP-Seq 82 analyses using human LNCaP cells (GSE83860) and found AR binding at the IL-8 83 promoter in the presence of the potent androgen dihydrotestosterone (DHT; Fig. 1e top). 84This androgen dependent binding was verified by ChIP-qRT-PCR ( Fig. 1f). 85To further explore the role of AR in IL-8 regulation, we interrogated RNA polymerase 86 binding and transcription marks found at sites of active promoters 20 . In the presence of 87 DHT, binding of RNA polymerase II (pol II), phosphorylated serine 2 RNA polymerase II 88 5 (pSer2 pol II) and histone H3 tri-methyl Lys4 (H3K4me3) to the IL-8 locus were 89 substantially reduced, consistent with reduced transcriptional activity ( Fig. 1f). 90Conversely, pSer2 pol II binding to the promoter of the well-established AR-regulated 91 gene PSA (KLK3), was significantly increased in the presence of DHT as expected 92 (Extended Data Fig. 1e). Consistent with a role for inflammation, TNFα significantly 93 increased p65 binding at the IL-8 (CXCL8) promoter in LNCaP cells (Fig. 1e bottom). No 94 significant binding of AR was detected at the promoters of the chemokines CXCL1, 95 CXCL2, CXCL5 or CXCL12 (Extended Data Fig. 1f). These data suggest that AR directly 96 suppresses IL-8 expression through repressive AR binding to the IL-8 promoter. Taken 97 together, we found that IL-8 transcription is up-regulated by pro-inflammatory signaling, 98 and down-regulated by AR signaling (Fig. 1g). 99 We next investigated the effects of ADT on the expression of Cxcl15 in vivo, using RNA 1...
DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.
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