A PRC1–RNF2 knockout punch for cancer

Shedding light on epigenetic mechanisms controlling anti-tumor immune responses, a new study shows that the tumor-intrinsic ring finger protein 2 (RNF2), the catalytic subunit of Polycomb repressor complex 1 (PRC1), acts as a negative regulator of a collaborative NK and CD4+ T cell anti-tumor immune response against breast cancer.


TUMOR IMMUNOLOGY
A PRC1-RNF2 knockout punch for cancer Shedding light on epigenetic mechanisms controlling anti-tumor immune responses, a new study shows that the tumor-intrinsic ring finger protein 2 (RNF2), the catalytic subunit of Polycomb repressor complex 1 (PRC1), acts as a negative regulator of a collaborative NK and CD4 + T cell anti-tumor immune response against breast cancer.
Janneke G. C. Peeters and Michel DuPage S everal tumor-intrinsic factors have been reported to modulate the composition of the tumor microenvironment (TME) and thus the anti-tumor immune response 1 . Tumor-intrinsic epigenetic regulators are of particular interest because of their druggable nature and the natural plasticity of epigenetic modifications. Inhibitors of the histone methyltransferase enhancer of zeste homolog 2 (EZH2), a member of Polycomb repressive complex 2 (PRC2), have recently been approved to treat cancers with hyperactivated EZH2 2 . Interestingly, preclinical studies indicate that EZH2 inhibition in cancer cells may also enhance the immune response against the tumor 3 . The genes encoding subunits of PRC1 are typically not mutated in cancer, but their dysregulation or genetic amplification has been described for many cancer types, including breast cancer 4 . Recent findings indicate that amplification or overexpression of the PRC1 core factor RNF2 is associated with reduced overall survival in patients with triple-negative breast cancer (TNBC) 4,5 . In this issue of Nature Cancer, Zhang et al. 6 demonstrate that this association is likely the result of RNF2 functioning to prevent effective anti-tumor immunity. Deletion of Rnf2 in two preclinical mouse TNBC models (4T1 and EMT6 mammary tumor cells) led to durable immune-mediated tumor rejection by increasing the genomic accessibility and expression of immune-response-associated genes. This increased the infiltration, activation and function of both CD4 + T cells and NK cells in the TME, which collaboratively controlled the tumor. From an epigenetic and immunological perspective, the authors' findings have several far-reaching implications for the treatment of TNBC.
Rnf2 encodes the protein RNF2/RING1B, which functions as an E3 ubiquitin ligase within PRC1. RNF2/RING1B mediates monoubiquitylation of lysine 119 of histone H2A (H2AK119Ub), a modification associated with gene repression 7 . Zhang et al. 6 showed by immunoprecipitation of RNF2 in TNBC cells that RNF2 predominantly interacts with subunits of canonical PRC1 (cPRC1). Indeed, disruption of Bmi1, encoding another cPRC1 complex subunit, or of the downstream H2AK119Ub reader protein gene Rsf1 similarly disrupted tumor growth in immunocompetent mouse tumor models. ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) of Rnf2 KO cells revealed many genomic regions that became more accessible, but also many that were less accessible. This fits with recent reports demonstrating that PRC1 can play opposing roles in regulating chromatin accessibility not only by closing chromatin but, in certain contexts, by binding to and promoting the transcription of cancer-specific enhancers 4 . In estrogen-receptor-positive (ER + ) breast cancer, RNF2 functions as a cell-intrinsic suppressor of metastasis, and high levels of RNF2 are associated with improved survival 4 Fig. 1 | RNF2 blocks a collaborative NK and CD4 + T cell anti-tumor immune response. The immune response against triple-negative breast cancers expressing the canonical PRC1 complex subunits RNF2, BMI1 and the ubiquitin reader protein RSF1 engages CD8 + T cells, which alone are insufficient for tumor control. Disruption of either RNF2 (as illustrated), BMI1 or RSF1 leads to increased genomic accessibility and expression of immune-response-associated genes, including MHC class II genes. This leads to direct recognition and killing of tumor cells by CD4 + T cells and NK cells. Further, CD4 + T cells and NK cells both enhance each other's activity, in part by producing IFNγ.
associating with different transcriptional regulators in TNBC compared to ER + breast cancer 4 . Together, these findings suggest that the role of RNF2 in the regulation of breast cancer is highly context specific.
To test whether the E3 ligase activity of RNF2 was needed for its role in tumor control, Zhang et al. 6 developed a TNBC cell line containing a catalytically dead RNF2 mutant. Compared to control cells (that is, TNBC cells with intact RNF2), tumor growth was not affected in the TNBC cells containing mutant RNF2, indicating that suppression of the anti-tumor immune response was not dependent on RNF2's E3 ligase activity. This was surprising because RNF2 deletion in both 4T1 and EMT6 cells significantly reduced H2AK119Ub levels, and deletion of the H2AUb reader protein gene Rsf1 also led to tumor suppression. However, these findings are in line with previous studies in embryonic stem cells demonstrating that the ability of RNF2 to ensure a compact chromatin state does not require its histone ubiquitination activity 8,9 . Furthermore, RNF2 might not be the only critical histone H2A ubiquitin ligase in TNBC. RING1A was shown to be more enzymatically active toward histone H2A than RING1B in another TNBC cell line, the human line MDA-MB-231 4 . In addition, human breast cancer cell lines often exhibit amplification of chromosome locus 17q23, which contains TRIM37, encoding another E3 ubiquitin ligase for H2AK119 with oncogenic potential 10 . The existence of alternative ubiquitin ligases may explain the fact that Zhang et al. found that Rsf1 deletion was also critical for cancer immunity. Further investigation, for example through H2AK119Ub chromatin immunoprecipitation sequencing (ChIP-seq) in TNBC cell lines, will be required to determine the importance of H2AK119Ub in regulating the anti-tumor immune response.
Zhang et al. 6 demonstrated that the genomic regions that gained accessibility after Rnf2 KO were enriched in immune-related gene signatures and that the genes in these regions were more highly expressed. Among the genes upregulated and shown to be bound by RNF2 using ChIP were the MHC class II genes H2-Ab1 and H2-Eb1 4 . Knockout of H2-Ab1 and H2-Eb1 in Rnf2 KO 4T1 tumor cells restored tumor growth, indicating that RNF2 influences the anti-tumor response by preventing tumor cell direct recognition by CD4 + T cells. Tumor-directed cytotoxicity of CD4 + T cells has recently been described both in preclinical mouse models and in patient-derived CD4 + T cells 11 . Indeed, Zhang et al. found that CD4 + T cells displayed tumoricidal activity against Rnf2 KO tumor cells. In addition, increased recognition of tumor cells by CD4 + T cells also enhanced the anti-tumor NK cell response because CD4 + T cell depletion reduced NK cell infiltration, activation and function. Interestingly, NK cell depletion also affected the anti-tumor CD4 + T cell response, suggesting a cooperative role for NK cells and CD4 + T cells in the rejection of Rnf2 KO tumors. In vitro co-culture experiments showed mutual activation of CD4 + T cells and NK cells upon Rnf2 loss and indicated that this activation was driven by interferon-γ (IFNγ), but not interleukin 2, produced by CD4 + T cells and NK cells. In agreement with this, IFNγ-neutralizing antibodies restored the growth of Rnf2 KO tumors in vivo. Importantly, IFNγ did not act directly on tumor cells, for example by increasing MHC class II expression, because IFNγ receptor deletion in Rnf2 KO cells did not prevent immune-mediated cancer rejection. Finally, re-challenge experiments of cured mice showed that CD4 + T cells were uniquely required for anti-tumor memory responses. Similar interplay of NK cells and CD4 + T cells has been described before. For example, eradication of MHC-class-I-low tumors has been described to occur by NK cell-mediated killing of tumor cells, which results in the release of tumor antigens that can induce tumor-specific CD4 + T cells 12 . Similarly, Zhang et al. found that NK cells acted early to control tumor growth, and CD4 + T cells had a dominant role later in controlling tumors and establishing anti-tumor immune memory. Further experiments will be required to fully appreciate this dynamic T-NK crosstalk, for instance by identifying the additional factors mediating their communication, the location of these interactions (TME versus elsewhere) and the timing of the interactions during the anti-tumor response (Fig. 1).
Surprisingly, whereas CD4 + T cell and NK cell depletion both abrogated Rnf2 KO tumor control, CD8 + T cell depletion did not have a similar effect. This is an unexpected finding because CD8 + T cells have been demonstrated to be involved in the anti-tumor immune response against TNBC cell lines expressing RNF2, and because high numbers of CD8 + tumor-infiltrating lymphocytes are associated with better survival in TNBC 13 . The absence of CD8 + T cell control of Rnf2 KO tumors could be related to the downregulation of MHC class I proteins in Rnf2 KO cells, but this needs further exploration. Nevertheless, these results suggest that mutated or dysregulated chromatin modifiers can fundamentally alter the type of immunological response waged against cancer.
In view of translating the findings of Zhang et al. 6 into therapeutic approaches for cancer, the observation that the catalytic activity of RNF2 is dispensable for its effect on tumor growth is important. A small-molecule inhibitor of RNF2, PRT4165, exists that inhibits its E3 ubiquitin ligase activity 14 . However, based on this study, it is unlikely that an enzymatic inhibitor of RNF2 will have the desired effect. As an alternative approach, RNF2-containing enhancers in MDA MB-231 cells have been demonstrated to require the activity of bromodomain-containing protein 4 (BRD4), making BET inhibition an enticing strategy to explore in future studies 4 . Finally, the effect of Rnf2 disruption on the immune compartment needs to be considered. For instance, in mouse macrophages, in addition to H2A ubiquitination and chromatin compaction, RNF2 has been demonstrated to promote STAT1/STAT2 dissociation from DNA, thereby negatively regulating interferon-stimulated gene expression 15 . It is likely that RNF2 also plays important parts in NK cells and CD4 + T cells, and additional studies investigating how RNF2 disruption affects the activation and function of distinct immune cell populations are needed. In conclusion, given that current immunotherapies are largely focused on enhancing the CD8 + T cell response, the work of Zhang